Exonuclease Enabled Proximity Extension Assays

ABSTRACT

The present invention relates to a proximity probe based detection assay (“proximity assay”) for an analyte in a sample, specifically a proximity probe extension assay (PEA), an in particular to an improvement in the method to reduce non-specific “background” signals, wherein the improvement comprises the use in such assays of a component comprising 3′ exonuclease activity, said method comprising: (a) contacting said sample with at least one set of at least first and second proximity probes, which probes each comprise an analyte-binding domain and a nucleic acid domain and can simultaneously bind to the analyte; (b) allowing the nucleic acid domains of the proximity probes to interact with each other upon binding of said proximity probes to said analyte, wherein said interaction comprises the formation of a duplex; (c) contacting said sample with a component comprising 3′ exonuclease activity; (d) extending the 3′ end of at least one nucleic acid domain of said duplex to generate an extension product, wherein the step may occur contemporaneously with or after step (c); and (e) amplifying and detecting the extension product.

The present invention relates to a proximity probe based detection assay(“proximity assay”) for an analyte in a sample, specifically a proximityprobe extension assay (PEA). In particular, the present inventionrelates to an improvement in the method to reduce non-specific“background” signals which arise in all sample types, and can beparticularly problematic in complex biological samples. The improvementcomprises the use in such assays of a component comprising 3′exonuclease activity.

In proximity probe extension assays proximity probes are used, whichbind to the analyte and have nucleic acid domains (or tags), thatinteract in a proximity-dependent manner upon said analyte binding. Theinteraction, generally via the formation of one or more nucleic acidduplexes (through the hybridisation of the nucleic acid domains),enables at least one of the nucleic acid domains to be extended from its3′ end. This extension product forms a detectable, preferablyamplifiable, nucleic acid detection product, or detection tag, by meansof which said analyte may be detected.

In the present invention the component comprising 3′ exonucleaseactivity is added before or contemporaneously with component necessaryfor the extension of the nucleic acid domain(s), e.g. a polymeraseenzyme. Thus, it is believed that the component comprising 3′exonuclease activity is able to degrade nucleic acid domains with a freeand unprotected 3′ end, i.e. domains on proximity probes that are notbound to the target analyte and therefore do not form a specificinteraction or duplex. In the present invention the observable effect ofthe inclusion of a component comprising 3′ exonuclease activity is toprevent the production of non-specific extension products fromnon-hybridised or “non-duplexed” nucleic acid domains, therebyincreasing both the specificity and sensitivity of the assay. Thepresent invention also provides a kit comprising a component comprising3′ exonuclease activity for use in the methods of the invention.

In general, a proximity assay relies on the principle of “proximityprobing”, wherein an analyte is detected by the binding of multiple(i.e. two or more, generally two or three) probes, which when broughtinto proximity by binding to the analyte (hence “proximity probes”)allow a signal to be generated. Typically, at least one of the proximityprobes comprises a nucleic acid domain (or moiety) linked to theanalyte-binding domain (or moiety) of the probe, and generation of thesignal involves an interaction between the nucleic acid moieties and/ora further functional moiety which is carried by the other probe(s). Thussignal generation is dependent on an interaction between the probes(more particularly between the nucleic acid or other functionalmoieties/domains carried by them) and hence only occurs when both thenecessary (or more) probes have bound to the analyte, thereby lendingimproved specificity to the detection system. The concept of proximityprobing has been developed in recent years and many assays based on thisprinciple are now well known in the art. For example, “proximity probeextension assays” or “proximity extension assays” (PEA) refer to aspecific type of assay, which utilises the extension of a nucleic aciddomain, i.e. the templated addition of nucleotides to the end of anucleic acid molecule, to generate a detectable signal.

Proximity-probe based detection assays, and particularly proximityextension assays permit the sensitive, rapid and convenient detection orquantification of one or more analytes in a sample by converting thepresence of such an analyte into a readily detectable or quantifiablenucleic acid-based signal, and can be performed in homogeneous orheterogeneous formats.

Proximity probes of the art are generally used in pairs, andindividually consist of an analyte-binding domain with specificity tothe target analyte, and a functional domain, e.g. a nucleic acid domaincoupled thereto. The analyte-binding domain can be for example a nucleicacid “aptamer” (Fredriksson et al (2002) Nat Biotech 20:473-477) or canbe proteinaceous, such as a monoclonal or polyclonal antibody (Gullberget al (2004) Proc Natl Acad Sci USA 101:8420-8424). The respectiveanalyte-binding domains of each proximity probe pair may havespecificity for different binding sites on the analyte, which analytemay consist of a single molecule or a complex of interacting molecules,or may have identical specificities, for example in the event that thetarget analyte exists as a multimer. When a proximity probe pair comeinto close proximity with each other, which will primarily occur whenboth are bound to their respective sites on the same analyte molecule,the functional domains (e.g. nucleic acid domains) are able to interact.In the context of the present invention, for example, nucleic aciddomains may contain regions of complementarity to each other and hencethe nucleic acid domains of the proximity probe pair may hybridise toform a duplex. One or more of the domains may be extended to form a newnucleic acid sequence, generally by means of a polymerisation reactiontemplated by the nucleic acid domain of the other proximity probe. Thenew nucleic acid sequence thereby generated serves to report thepresence or amount of analyte in a sample, and can be qualitatively orquantitatively detected, for example by realtime, quantitative PCR(q-PCR).

Many variations of proximity probe based assays exist. For example,proximity extension assays are described in WO 01/61037, U.S. Pat. No.6,511,809 and WO 2006/137932 and both heterogeneous (i.e. the analyte isfirst immobilised to a solid substrate by means of a specificanalyte-binding reagent) and homogeneous (i.e. in solution) formats forproximity probe based assays have been disclosed, e.g. WO 01/61037, WO03/044231, WO 2005/123963, Fredriksson et al (2002) Nat Biotech20:473-477 and Gullberg et al (2004) Proc Natl Acad Sci USA101:8420-8424.

Although pairs of proximity probes are generally used, modifications ofthe proximity-probe detection assay have been described, in e.g. WO01/61037, WO 2005/123963 and WO 2007/107743, where three proximityprobes are used to detect a single analyte molecule. For example, thethird proximity probe may be used to provide a further nucleic aciddomain that can interact with nucleic acid domains of the first twoproximity probes.

In addition to modification to the proximity-probe detection assay,modifications of the structure of the proximity probes themselves havebeen described, in e.g. WO 03/044231, where multivalent proximity probesare used. Such multivalent proximity probes comprise at least two, butas many as 100, analyte-binding domains conjugated to at least one, andpreferably more than one, nucleic acid(s).

Proximity-probe based detection assays and particularly proximityextension assays, have proved very useful in the specific and sensitivedetection of proteins in a number of different applications, e.g. thedetection of weakly expressed or low abundance proteins. However, suchassays are not without their problems and room for improvement exists,with respect to both the sensitivity and specificity of the assay.

The sensitivity of the conventional proximity assays, e.g. proximityextension assays, as described above, is limited by two main factors:(i) the affinity of the analyte-binding domains for the target analyteand (ii) the non-specific background signal arising from the randomproximity of non-bound probes, particularly probe pairs. Using probeshaving binding domains with high affinity for the analyte, sensitivityis limited to the detection of approximately 6000 molecules.Traditionally, in order to achieve a low level of background, very lowconcentrations of proximity probes must be used. This precludes anyattempt to compensate for probes comprising low affinity analyte-bindingdomains by using higher concentrations of probe. It has therefore beenfound that this may limit the sensitivity of the assay, and the rangeover which quantitative results may be obtained.

Other methods for reducing non-specific background signal have beenproposed, such as using blocking reagents, e.g. blockingoligonucleotides, which bind to the free ends of the nucleic aciddomains on the proximity probes until displaced by, e.g. a displaceroligonucleotide. The addition of a displacer oligonucleotide after theproximity probes have been allowed to bind to the analyte means thatinteraction of the nucleic acid domains of the proximity probes islikely to occur only for the proximity probes bound to the targetanalyte. Further methods for reducing background signal have centred onimproving the detection of the nucleic acid extension product.

However, there is still room to improve the level of background signaland in order to overcome the limitations of the proximity assay,particularly proximity extension assays known in the art, as describedabove, it has now been found that the use of a component comprising 3′exonuclease activity, before or contemporaneously with the extensionreaction, significantly improves the sensitivity and specificity of theassay. Preferably the component comprising 3′ exonuclease activity iscontacted with the sample at the same time as the component used for theextension of the nucleic acid domains, e.g. a polymerase enzyme. In aparticularly preferred embodiment, the polymerase used to extend thenucleic acid domains comprises 3′ exonuclease activity.

By including in the assay a component that is capable of degradingnon-proximal nucleic acid domains (domains with a free and unprotected3′ end), i.e. degrading nucleic acid domains that have not formed a(stable) sequence specific duplex with a nucleic acid domain of aproximity probe bound to the same target analyte, it is possible toreduce the non-specific background signal present in the assay.Alternatively put, the use of a component comprising 3′ exonucleaseactivity may be seen as reducing or preventing the production ofunwanted, undesired or non-specific extension products.

Whilst the use of components comprising 3′ exonuclease activity is wellknown in methods used for producing nucleic acid molecule extensionproducts, the use of said activity in methods for detecting an analytein a sample, particularly in the proximity extension assays of thepresent invention provides a unique and unexpected advantage overprevious proximity probe based assays.

In general, components of proximity probe based assays are selectedspecifically to avoid the degradation of other components in the assay.Degrading, destroying or corrupting part or all of the proximity probes,particularly the nucleic acid domains, would be expected to impede orhamper the production of the nucleic acid molecule that acts as thesignal to indicate the presence of the target analyte, for example, byreducing the concentration of potential interacting partners.

Furthermore, the presence of such an activity may be seen as an obstacleto the further amplification of the nucleic acid product, i.e. saidactivity would need to be removed to avoid degradation of the signalnucleic acid molecule or components required for the amplificationreaction. The addition of further steps to an analyte detection assay isgenerally undesirable because it reduces the simplicity of the methodthereby increasing the difficulty of automation and/or adaptation tohigh throughput use. Similarly, the addition of components to the assayincreases the complexity of the sample, which may be expected to reducethe sensitivity of the assay.

The method of the present invention, however, relies on the superiorreduction in background (i.e. an increase in the signal:noise ratio)seen when a component comprising 3′ exonuclease activity is contactedwith the sample in a PEA. In particular the 3′ exonuclease activity isadvantageously supplied as part of the nucleic acid polymerase used toextend the nucleic acid domains of the proximity probes. Alternativelyor additionally, the component comprising 3′ exonuclease activity may beprovided as an independent component (i.e. a separate entity not coupledto, or part of, a polymerase enzyme). Thus it can be seen that thecomponent comprising 3′ exonuclease activity can be contacted with thesample in any appropriate form.

Whilst not wishing to be bound by theory, it is theorised that the 3′exonuclease activity is able to degrade the nucleic acid domains ofunbound proximity probes with a free and unprotected 3′ end (e.g. wherethe nucleic acid domain is coupled to the analyte-binding domain by its5′ end and where the “free” 3′ end is not modified to be resistant to 3′exonuclease activity). In this respect, only duplexes formed between thenucleic acid domains of proximity probes that are both bound to thetarget analyte may form a stable interaction (under the conditions ofthe assay, the proximity probes are not easily dissociated from thetarget analyte once bound, thereby enabling a steady, i.e. non-transientor lasting, duplex to be formed). The formation of a duplex acts toprotect the nucleic acid domain of the proximity probe from the 3′exonuclease activity. However, proximity probes that are not bound tothe target analyte cannot form a stable duplex, i.e. free/unbound probes(not bound to the target analyte) may interact with the nucleic aciddomains of other proximity probes or components in the sample onlytransiently (forming an unstable and temporary interaction or duplex).Hence, at any one time the unbound probes of the assay are likely to bein a non-hybridised state and those unbound probes comprising a nucleicacid domain with a free and unprotected 3′ end will be a substrate forthe 3′ exonuclease component. The degradation of the nucleic aciddomains of these unbound probes prevents the production of non-specificextension products that might otherwise arise in an assay where unboundprobes are allowed to persist, e.g. due to extension products arisingfrom duplexes formed by transient or non-specific interactions withnucleic acid domains of proximity probes or other components in thesample.

It is the addition of this 3′ exonuclease component that results in animproved reduction in non-specific background signal in aproximity-probe extension assay. As shown in more detail in theExamples, the present invention represents a significant advance overthe proximity extension assays known in the art. Surprisingly, it isshown that a reduction in non-specific background activity is observedin the methods of the invention which use a component comprising 3′exonuclease activity, even in samples that comprise already high levelsof exonuclease activity, e.g. plasma. The addition of the 3′ exonucleasecomponent results in a similar improvement in the signal:noise ratio inassays performed using samples containing target analyte in both bufferand plasma, which was entirely unexpected. The consequence of such areduction in background signal is an increase in both the specificityand sensitivity of the proximity-probe detection assays.

Moreover, the methods disclosed herein also include a simplified PEA,wherein the components used to amplify the extension product can becombined with the components of the extension assay prior to theinactivation of the component comprising 3′ exonuclease. As discussedbelow, the component comprising 3′ exonuclease activity would beexpected to degrade certain components of the amplification reaction,e.g. PCR primers, thereby necessitating the addition of these componentsonly after the 3′ exonuclease activity has been inactivated. However,the methods disclosed herein describe the modification of variouscomponents that enable the reagents of both stages of the assay to bepre-mixed. Advantageously, this results in fewer pipetting steps andless hands-on time, which allows easy automation of the assay andreduction in error.

Accordingly, the invention can be seen to provide the use of a componentcomprising 3′ exonuclease activity in a proximity-probe extension assayto detect an analyte in a sample, wherein the extension product issubsequently amplified and detected.

More particularly, the component comprising 3′ exonuclease activity isused during the step of generating an extension product in theproximity-probe extension assay, and put more specifically during thestep of conducting a polymerase-catalysed extension reaction to generatean extension product following interaction of the nucleic acid domainsof proximity probes used in the proximity assay (such interactiongenerally being hybridisation of the nucleic acid domains, andsubsequent extension of at least one domain; generally speaking thenucleic acid domains hybridise such that one nucleic acid domain maytemplate the extension of another domain). Thus, the component having 3′exonuclease activity is included in the step of generating the initialextension product in the proximity-probe based extension assay, or inother words the step of generating an extension product from interactionof the nucleic acid domains of proximity probes in a proximity probeextension assay.

Alternatively viewed, this aspect of the invention provides a method ofdetecting an analyte in a sample, which method comprises aproximity-probe extension assay, wherein said assay comprises the use ofa component comprising 3′ exonuclease activity in the extension step ofthe assay, and the extension product is subsequently amplified anddetected.

Viewed from yet another aspect, the invention provides a method ofreducing non-specific extension products (or improving the signal:noiseratio) in a proximity-probe extension assay for detecting an analyte ina sample, said method comprising including in the extension step of saidassay a component comprising 3′ exonuclease activity, wherein thespecific extension product is subsequently amplified and detected.

As noted above, the extension step of a proximity probe extension assayis the step of generating the initial extension product which issubsequently detected as a means of detecting the analyte. In otherwords, the extension step is the step of generating an extension productbased on interaction of the nucleic acid domains of proximity probes,specifically hybridisation of the nucleic acid domains and subsequentextension of at least one of the domains, for example using the other astemplate.

The method of the invention is, and the component comprising 3′exonuclease activity is for use in, a proximity-probe based assay, andthe probe is a proximity probe. The proximity probe based assay may beany of the assays known in the art, for example as described above,which use proximity probes to detect an analyte in a sample.Specifically, the assay is a proximity extension assay, being based onthe detection of interactions between the nucleic acid domains ofproximity probes by hybridisation, and extension of one or more of thosedomains.

Accordingly, in one preferred aspect the present invention provides amethod for detecting an analyte in a sample, comprising:

(a) contacting said sample with at least one set of at least first andsecond proximity probes, which probes each comprise an analyte-bindingdomain and a nucleic acid domain and can simultaneously bind to theanalyte;

(b) allowing the nucleic acid domains of the proximity probes tointeract with each other upon binding of said proximity probes to saidanalyte, wherein said interaction comprises the formation of a duplex;

(c) contacting said sample with a component comprising 3′ exonucleaseactivity;

(d) extending the 3′ end of at least one nucleic acid domain of saidduplex to generate an extension product, wherein the step may occurcontemporaneously with or after step (c); and

(e) amplifying and detecting the extension product.

As described in more detail below, the analyte-binding domain may bindthe analyte directly or indirectly.

It will be seen therefore that the component comprising 3′ exonucleaseactivity may be used to extend the sensitivity and/or specificity of aproximity extension assay. The methods of the invention may thus beconsidered as methods of increasing the signal:noise ratio of proximityextension assay (for an analyte). Expressed another way, the methods ofthe present invention may be used to reduce the amount of non-specificextension products in a proximity extension assay. Alternatively, theinvention may be viewed as providing a method of disrupting orinhibiting the formation of non-specific duplexes in proximity extensionassays. Yet further still, the invention may be seen to provide a methodof degrading the nucleic acid domain of unbound proximity probes inproximity extension assays.

As described briefly above, proximity extension assays concern theextension of at least one nucleic acid domain following the formation ofa duplex between the nucleic acid domains of two or more proximityprobes, when said probes are bound to the target analyte. However, saidduplex (the hybridisation of two complementary nucleic acid domains) maybe formed in many ways, depending on the orientation of the nucleic aciddomain on the proximity probe.

A representative sample of proximity extension assay formats is shownschematically in FIG. 1 and these embodiments are described in detailbelow. However, these different “versions” of PEA are in no way intendedto be limiting on the scope of the invention. Other permutations wouldbe apparent to the skilled person from the below description and areintended to be encompassed by the present invention. In essence, thepresent invention requires simply that at least one of the proximityprobes (which is not limited to a proteinaceous molecule or an antibody,as shown, although this is a preferred aspect of the invention) has anucleic acid domain comprising a free 3′ end that is capable of beingextended, and wherein said extension may be templated by the nucleicacid domain of a second proximity probe. In this regard, and asdescribed in more detail below, the nucleic acid domains of theproximity probes may be single-stranded or partially double stranded,but are configured such that single stranded regions of the domains areavailable for interaction with each other by hybridisation. The nucleicacid domains of respective proximity probes may also interact by eachhybridising to a common “splint” nucleic acid molecule, therebyindirectly forming a duplex. Thus in step (b) of the method set outabove, the interaction of the nucleic acid domains to form a duplex maybe direct or indirect; the nucleic acid domains which are attached tothe analyte-binding domains of the proximity probes may hybridise toeach other to form a duplex directly or they may form a duplexindirectly by each hybridising to a further nucleic acid molecule. Sucha further nucleic acid molecule may be regarded as the second strand ofa partially double stranded nucleic acid domain; it will hybridise to atleast a region of a nucleic acid molecule attached to the analytebinding domain of a proximity probe (thereby forming a partially doublestranded nucleic acid domain which is attached via one strand thereof),leaving a terminal (e.g. 3′) single stranded region which iscomplementary to a region of the nucleic acid domain of anotherproximity probe, and which is therefore available for interaction byhybridisation with the nucleic acid domain of said other proximityprobe. Alternatively the “splint” may be provided as the nucleic aciddomain of a further proximity probe.

Version 1 of FIG. 1 depicts a “conventional” proximity extension assay,wherein the nucleic acid domain (shown as an arrow) of each proximityprobe is attached to the analyte-binding domain (shown as an inverted“Y”) by its 5′ end, thereby leaving two free 3′ ends. When saidproximity probes bind to their respective analyte-binding targets on theanalyte (the analyte is not shown in the figure) the nucleic aciddomains of the probes, which are complementary at their 3′ ends, areable to interact by hybridisation, i.e. form a duplex. The addition of,e.g. a nucleic acid polymerase enzyme, allows each nucleic acid domainto be extended using the nucleic acid domain of the other proximityprobe to template that extension. In accordance with the methods of theinvention the extension products may be specifically amplified anddetected, thereby detecting the target analyte.

Version 2 of FIG. 1 depicts an alternative proximity extension assay,wherein the nucleic acid domain of the first proximity probe is attachedto the analyte-binding domain by its 5′ end and the nucleic acid domainof the second proximity probe is attached to the analyte-binding domainby its 3′ end. The nucleic acid domain of the second proximity probetherefore has a free 5′ end (shown as a blunt arrow), which cannot beextended using a typical nucleic acid polymerase enzyme (which extendonly 3′ ends). The 3′ end of the second proximity probe is effectively“blocked”, i.e. it is not “free” and it cannot be extended because it isconjugated to, and therefore blocked by, the analyte-binding domain. Inthis embodiment, when the proximity probes bind to their respectiveanalyte-binding targets on the analyte, the nucleic acid domains of theprobes, which share a region of complementary at their 3′ ends, are ableto interact by hybridisation, i.e. form a duplex. However, in contrastto version 1, only the nucleic acid domain of the first proximity probe(which as a free 3′ end) may be extended using the nucleic acid domainof the second proximity probe as a template. As above, the extensionproduct may be amplified and detected, thereby detecting the targetanalyte.

In version 3 of FIG. 1, like version 2, the nucleic acid domain of thefirst proximity probe is attached to the analyte-binding domain by its5′ end and the nucleic acid domain of the second proximity probe isattached to the analyte-binding domain by its 3′ end. The nucleic aciddomain of the second proximity probe therefore has a free 5′ end (shownas a blunt arrow), which cannot be extended. However, in thisembodiment, the nucleic acid domains which are attached to the analytebinding domains of the respective proximity probes do not have regionsof complementarity and therefore are unable to form a duplex directly.Instead, a third nucleic acid molecule is provided that has a region ofhomology with the nucleic acid domain of each proximity probe, whichacts as a “molecular bridge” or a “splint” between the nucleic aciddomains. This “splint” oligonucleotide bridges the gap between thenucleic acid domains, allowing them to interact with each otherindirectly, i.e. each nucleic acid domain forms a duplex with the splintoligonucleotide. Thus, when the proximity probes bind to theirrespective analyte-binding targets on the analyte, the nucleic aciddomains of the probes each interact by hybridisation, i.e. form aduplex, with the splint oligonucleotide. It can be seen therefore thatthe third nucleic acid molecule or splint may be regarded as the secondstrand of a partially double stranded nucleic domain provided on one ofthe proximity probes. For example, one of the proximity probes may beprovided with a partially double-stranded nucleic acid domain, which isattached to the analyte binding domain via the 3′ end of one strand andin which the other (non-attached) strand has a free 3′ end. Thus such anucleic acid domain has a terminal single stranded region with a free 3′end. In this embodiment the nucleic acid domain of the first proximityprobe (which as a free 3′ end) may be extended using the “splintoligonucleotide” (or single stranded 3′ terminal region of the othernucleic acid domain) as a template and may advantageously be ligated tothe 5′ end of the nucleic acid domain (specifically the 5′ end of theattached strand, or alternatively put the 5′ end of the double-strandedportion of the nucleic acid domain) of the second proximity probe.Alternatively or additionally, the free 3′ end of the splintoligonucleotide (i.e. the unattached strand, or the 3′ single-strandedregion) may be extended using the nucleic acid domain of the firstproximity probe as a template. As above, the extension products may beamplified and detected, thereby detecting the target analyte.

As is apparent from the above description, in one embodiment, the splintoligonucleotide may be provided as a separate component of the assay. Inother words it may be added separately to the reaction mix (i.e. addedseparately to the proximity probes to the sample containing theanalyte). Notwithstanding this, since it hybridises to a nucleic acidmolecule which is part of a proximity probe, and will do so upon contactwith such a nucleic acid molecule, it may nonetheless be regarded as astrand of a partially double-stranded nucleic acid domain, albeit thatit is added separately. Alternatively, the splint may be pre-hybridisedto one of the nucleic acid domains of the proximity probes, i.e.hybridised prior to contacting the proximity probe with the sample. Inthis embodiment, the splint oligonucleotide can be seen directly as partof the nucleic acid domain of the proximity probe, i.e. wherein thenucleic acid domain is a partially double stranded nucleic acidmolecule, e.g. the proximity probe may be made by linking a doublestranded nucleic acid molecule to an analyte-binding domain (preferablythe nucleic acid domain is conjugated to the analyte-binding domain by asingle strand) and modifying said nucleic acid molecule to generate apartially double stranded nucleic acid domain (with a single strandedoverhang capable of hybridising to the nucleic acid domain of the otherproximity probe). Hence, the extension of the nucleic acid domain of theproximity probes as defined herein encompasses also the extension of the“splint” oligonucleotide. Advantageously, when the extension productarises from extension of the splint oligonucleotide, the resultantextended nucleic acid strand is coupled to the proximity probe pair onlyby the interaction between the two strands of the nucleic acid molecule(by hybridisation between the two nucleic acid strands). Hence, in theseembodiments, the extension product may be dissociated from the proximityprobe pair using denaturing conditions, e.g. increasing the temperature,decreasing the salt concentration etc. This is particularly useful in aheterogeneous format, wherein the target analyte is bound to a solidsubstrate, because the extension products can be separated easily fromother components of the assay, e.g. the proximity probes bound to theimmobilised analyte may be in the solid phase, whereas the extensionproduct, following denaturation, may be in the liquid phase.

Whilst the splint oligonucleotide depicted in Version 3 of FIG. 1 isshown as being complementary to the full length of the nucleic aciddomain of the second proximity probe, this is merely an example and itis sufficient for the splint to be capable of forming a duplex with theends (or near the ends) of the nucleic acid domains of the proximityprobes, i.e. to bridge the gap, as defined further below.

In another embodiment, the splint oligonucleotide may be provided as thenucleic acid domain of a third proximity probe as described in WO2007/107743, which is incorporated herein by reference, whichdemonstrates that this can further improve the sensitivity andspecificity of proximity probe assays.

Version 4 of FIG. 1, is a modification of Version 1, wherein the nucleicacid domain of the first proximity probe comprises at its 3′ end asequence that is not fully complementary to the nucleic acid domain ofthe second proximity probe. Thus, when said proximity probes bind totheir respective analyte-binding targets on the analyte the nucleic aciddomains of the probes are able to interact by hybridisation, i.e. form aduplex, but the extreme 3′ end of the nucleic acid domain (the part ofthe nucleic acid molecule comprising the free 3′ hydroxyl group) of thefirst proximity probe is unable to hybridise and therefore exists as asingle stranded, unhybridised, “flap”. On the addition of, e.g. anucleic acid polymerase enzyme, only the nucleic acid domain of thesecond proximity probe may be extended using the nucleic acid domain ofthe first proximity probe to template that extension. As discussedabove, the extension product may be specifically amplified and detected,thereby detecting the target analyte.

In this embodiment, it may be beneficial to modify one or morenucleotides at the 3′ end of the nucleic acid domain of the firstproximity probe, e.g. to be resistant to 3′ exonuclease activity.Suitable modifications are described further below. If the nucleic aciddomain is resistant to 3′ exonuclease activity, then only the nucleicacid domain of the second proximity probe may be extended. In contrast,if the nucleic acid domain is susceptible to 3′ exonuclease activity,then the “flap” may be degraded resulting in a 3′ end that is fullyhybridised (annealed) to the nucleic acid domain of the second proximityprobe. Hence, the nucleic acid domain of the first proximity probe alsomay be extended using the nucleic acid domain of the second proximityprobe as a template.

The final embodiment depicted in FIG. 1, namely Version 5, could beviewed as a modification of Version 3. However, in contrast to Version3, the nucleic acid domains of both proximity probes are attached totheir respective analyte-binding domains by their 5′ ends. In thisembodiment the 3′ ends of the nucleic acid domains are not complementaryand hence the nucleic acid domains of the proximity probes cannotinteract or form a duplex directly. Instead, a third nucleic acidmolecule is provided that has a region of homology with the nucleic aciddomain of each proximity probe which acts as a “molecular bridge” or a“splint” between the nucleic acid domains. This “splint” oligonucleotidebridges the gap between the nucleic acid domains, allowing them tointeract with each other indirectly, i.e. each nucleic acid domain formsa duplex with the splint oligonucleotide. Thus, when the proximityprobes bind to their respective analyte-binding targets on the analyte,the nucleic acid domains of the probes each interact by hybridisation,i.e. form a duplex, with the splint oligonucleotide. In accordance withVersion 3, it can be seen therefore that the third nucleic acid moleculeor splint may be regarded as the second strand of a partially doublestranded nucleic domain provided on one of the proximity probes. In apreferred example, one of the proximity probes may be provided with apartially double-stranded nucleic acid domain, which is attached to theanalyte binding domain via the 5′ end of one strand and in which theother (non-attached) strand has a free 3′ end. Thus such a nucleic aciddomain has a terminal single stranded region with at least one free 3′end. In this embodiment the nucleic acid domain of the second proximityprobe (which as a free 3′ end) may be extended using the “splintoligonucleotide” as a template. Alternatively or additionally, the free3′ end of the splint oligonucleotide (i.e. the unattached strand, or the3′ single-stranded region of the first proximity probe) may be extendedusing the nucleic acid domain of the second proximity probe as atemplate. As above, the extension products may be amplified anddetected, thereby detecting the target analyte.

As discussed above on connection with Version 3, the splintoligonucleotide may be provided as a separate component of the assay. Onthe other hand, since it hybridises to a nucleic acid molecule which ispart of a proximity probe, and will do so upon contact with such anucleic acid molecule, it may be regarded as a strand of a partiallydouble-stranded nucleic acid domain, albeit that it is added separately.Alternatively, the splint may be pre-hybridised to one of the nucleicacid domains of the proximity probes, i.e. hybridised prior tocontacting the proximity probe with the sample. In this embodiment, thesplint oligonucleotide can be seen directly as part of the nucleic aciddomain of the proximity probe, i.e. wherein the nucleic acid domain is apartially double stranded nucleic acid molecule, e.g. the proximityprobe may be made by linking a double stranded nucleic acid molecule toan analyte-binding domain (preferably the nucleic acid domain isconjugated to the analyte-binding domain by a single strand) andmodifying said nucleic acid molecule to generate a partially doublestranded nucleic acid domain (with a single stranded overhang capable ofhybridising to the nucleic acid domain of the other proximity probe).Hence, the extension of the nucleic acid domain of the proximity probesas defined herein encompasses also the extension of the “splint”oligonucleotide. Advantageously, when the extension product arises fromextension of the splint oligonucleotide, the resultant extended nucleicacid strand is coupled to the proximity probe pair only by theinteraction between the two strands of the nucleic acid molecule (byhybridisation between the two nucleic acid strands). Hence, in theseembodiments, the extension product may be dissociated from the proximityprobe pair using denaturing conditions, e.g. increasing the temperature,decreasing the salt concentration etc. This is particularly useful in aheterogeneous format, wherein the target analyte is bound to a solidsubstrate, because the extension products can be separated easily fromother components of the assay, e.g. the proximity probes bound to theimmobilised analyte may be in the solid phase, whereas the extensionproduct, following denaturation, may be in the liquid phase.

Whilst the splint oligonucleotide depicted in Version 5 of FIG. 1 isshown as being complementary to the full length of the nucleic aciddomain of the first proximity probe, this is merely an example and it issufficient for the splint to be capable of forming a duplex with theends (or near the ends) of the nucleic acid domains of the proximityprobes, i.e. to bridge the gap, as defined further below.

In another embodiment, the splint oligonucleotide may be provided as thenucleic acid domain of a third proximity probe as described in WO2007/107743, which is incorporated herein by reference, whichdemonstrates that this can further improve the sensitivity andspecificity of proximity probe assays.

From the above it will be understood that there are multiplepermutations of proximity extension assays, which all rely on theformation of a nucleic acid duplex comprising an extensible 3′ end (fortemplated extension) on the interaction between two (or more) proximityprobes, when such probes are bound to the analyte. The interactionbetween the probes (or more specifically, between their respectivenucleic acid domains, which includes splint oligonucleotides) is thusproximity-dependent; the binding of the detection probes, together, onthe analyte brings them into proximity, such that they (or moreparticularly, their nucleic acid domains) may interact. Accordingly, bydetecting the interaction (or the extension products generatedtherefrom), the analyte may be detected. In the method of the invention,the proximity probes may interact by hybridisation to each other(directly or indirectly) to allow the extension of one or more nucleicacid molecules. This extension may result in the nucleic acid domains oftwo proximity probes being conjugated, or joined to one another, and asplint oligonucleotide (which may form part of the nucleic acid domainof a proximity probe) assists in or mediates this interaction(conjugation). The extension (and if applicable the conjugation) may bedetected by detecting the extension and/or conjugation product(interaction product).

Whilst not wishing to be bound by theory, it is believed that the methodof the invention relies upon the addition of a component comprising 3′exonuclease activity degrading the free and unprotected 3′ ends ofnucleic acid domains of unbound proximity probes. It is thought that thenucleic acid domains of said unbound probes would otherwise bindnon-specifically and/or transiently (temporarily) to other unboundproximity probes or components of the assay to generatenon-specific/background extension products, thereby interfering with thedetection of the analyte.

Exonucleases are enzymes that work by cleaving nucleotides one at a timefrom the end of a polynucleotide chain by hydrolyzing phosphodiesterbonds at either the 3′ or the 5′ end. Thus exonucleases exist as either5′ or 3′ exonucleases, which nomenclature refers to the end from whichcleavage is initiated, i.e. 3′ exonucleases degrade nucleic acidmolecules in the 3′ to 5′ direction. Numerous types of exonucleaseenzyme are known to exist, which degrade DNA and/or RNA and may act ondouble stranded or single stranded nucleic acids. Whilst exonucleasesmay be distinct entities (separate enzymes with the sole function ofdegrading nucleic acids), e.g. a 5′ RNA exonuclease, which is found inboth eukaryotes and prokaryotes for the turnover of mRNA and exonucleaseI from E.coli, which degrades single stranded DNA in a 3′-5′ direction,much exonuclease activity arises from enzymes with multiple functions,e.g. many DNA polymerases comprise also 3′ and/or 5′ exonucleaseactivity (DNA polymerase I from E.coli).

Hence, a component comprising 3′ exonuclease activity for use in themethods of the invention includes any element that is capable ofdegrading a nucleic acid from its 3′ end. In a preferred aspect of theinvention, the component comprising 3′ exonuclease activity actspreferentially on single stranded nucleic acids, i.e. it has a greateractivity on single stranded molecules than double stranded, e.g. atleast 2, 3, 4, 5, 10, 20, 50 or 100 times more activity on singlestranded nucleic acid molecules.

The component comprising exonuclease activity must be capable ofdegrading, fully or partially, the nucleic acid domain of unboundproximity probes, i.e. probes not bound to the target analyte, whereinsaid nucleic acid domains have a free and unprotected 3′ end. Asdiscussed below, the nucleic acid domain of the proximity probes mayconsist of any nucleotide residues that are capable of participating inWatson-Crick type or analogous base pair interactions. However, inembodiments where the nucleic acid domains comprise DNA the componentcomprising 3′ exonuclease activity acts on DNA, and preferably has ahigher activity on DNA than RNA e.g. it has at least 2, 3, 4, 5, 10, 20,50 or 100 times more activity on DNA than RNA. Similarly, in embodimentswhere the nucleic acid domains comprise RNA the component comprising 3′exonuclease activity acts on RNA, and preferably has a higher activityon RNA than DNA e.g. it has at least 2, 3, 4, 5, 10, 20, 50 or 100 timesmore activity on RNA than DNA.

In one aspect of the invention, the component comprising 3′ exonucleaseactivity is an enzyme. In a particularly preferred embodiment the enzymeis a nucleic acid polymerase capable of extending the 3′ end of nucleicacid molecule that also comprises 3′ exonuclease activity. Specifically,the component comprising 3′ exonuclease activity may be selected fromany one or more of the group comprising T4 DNA polymerase, T7 DNApolymerase, Phi29 (Φ29) DNA polymerase, DNA polymerase I, Klenowfragment of DNA polymerase I, Pyrococcus furiosus (Pfu) DNA polymeraseand/or Pyrococcus woesei (Pwo) DNA polymerase. Hence, in someembodiments the component comprising 3′ exonuclease activity may be ahyperthermophilic enzyme, particularly a hyperthermophilic polymerasecomprising 3′ exonuclease activity, such as Pfu DNA polymerase or PwoDNA polymerase.

Hyperthermophilic enzymes are typically enzymes derived or obtained fromhyperthermophilic organisms, namely organisms that grow optimally atextreme temperatures above 90° C., for example at around 100° C. (ascompared with optimal growth at around 70° C., which is typically formost thermophiles). A hyperthermophilic polymerase (or more particularlya polymerase from a hyperthermophilic organism) may have optimumenzymatic activity at above normal biological temperatures, e.g. above37° C., such as above 40, 50, 60 or 70° C., typically above 60° C. or70° C. Significantly, such an enzyme will advantageously exhibit low, orreduced, activity at lower temperatures such as at room temperature,e.g. at 25° C. , or at 30° C. Particularly advantageously, polymeraseactivity may be low until a temperature of at least 45° C. or 50° C. isreached. Such enzymes have been identified from organisms that live inextreme temperature conditions, e.g. archaea (such as Pyrococcusfuriosus and Pyrococcus woesei etc). Enzymes of particular interest fromthese organisms are polymerase enzymes, which have been useful in thedevelopment of many molecular biology techniques, most notably PCR. Aswell as naturally occurring hyperthermophilic enzymes, thermostableenzymes with a high temperature optimum may be modified in order toconfer the property of reduced or low activity at room temperature, inorder to create an enzyme with the same or similar properties,specifically the same or similar temperature activity profile, as anaturally occurring hyperthermophilic enzyme, namely the combination ofa high temperature optimum (e.g. above 50, 55, or 60° C.) with low orreduced activity at room temperature (or at a lower temperature such as30° C. , 37° C. or 40° C.). Such modified polymerase enzymes include forexample so-called “hot start” derivatives of Taq polymerase, which e.g.gain activity at about 50° C. As used herein, the term“hyperthermophilic polymerase” includes not only naturally occurringenzymes but also all such modified derivatives, including alsoderivatives of naturally occurring hyperthermophilic polymerase enzymes.Particularly preferred hyperthermophilic enzymes for use in the methodsof the present application include Pfu DNA polymerase and Pwo DNApolymerase and derivatives, e.g. sequence-modified derivatives, ormutants thereof. Although in certain embodiments, a hyperthermophilicpolymerase may be advantageous or preferred, it is not necessary to usesuch an enzyme and in other embodiments any thermophilic or thermostablepolymerase may be used e.g. Taq polymerase (that it any polymeraseenzyme exhibiting stability at high temperatures, or an increasedtemperature optimum).

In another embodiment, the polymerase activity for extension of theproximity probe nucleic acid domain (and/or splint oligonucleotide) maybe provided by an enzyme with no or minimal 3′ exonuclease activity,e.g. the a subunit of DNA polymerase III from E.coli, the Klenow exo(−)fragment of DNA polymerase I, Taq polymerase, Pfu (exo⁻) DNA polymerase,Pwo (exo⁻) DNA polymerase etc., and the component comprising 3′exonuclease may be provided as a separate entity, e.g. another enzymecomprising 3′ exonuclease activity. Hence, the polymerase enzyme with noor minimal 3′ exonuclease activity may be a hyperthermophilic orthermostable polymerase. In a further embodiment, the componentcomprising 3′ exonuclease activity may be included in multiple forms,i.e. more than one component comprising 3′ exonuclease activity may beprovided, e.g. as part of the polymerase enzyme and as an independentenzyme that has as its primary function, 3′ exonuclease activity. In oneaspect of the invention, the component comprising 3′ exonucleaseactivity is exonuclease I.

Advantageously the component comprising 3′ exonuclease activity iscontacted with the sample before or contemporaneously with the componentrequired for the extension of the nucleic acid domain, but after thenucleic acid domains of the proximity probes have been allowed tointeract, i.e. to form a duplex. In this respect, the nucleic domains ofthe proximity probes must be at least partially single stranded oncontact with the sample such that they can interact with each other onbinding to the target analyte. Thus, if the component comprising 3′exonuclease activity was present (present in an active form) prior tothe formation of the aforementioned duplex, the nucleic acid domains ofall of the proximity probes with a free and unprotected 3′ end would besusceptible to degradation. In contrast, once the duplex has beenallowed to form between proximity probes bound to the target analyte,then only the nucleic acid domains of unbound proximity probes areavailable for degradation. As discussed above, probes not bound to thetarget analyte do not form a stable duplex, and whilst they may interactwith other non-bound probes or other components of the sample, theseinteractions are likely to be transient (temporary), thereby meaningthat the nucleic acid domains of these probes will be availablesubstrates for the component comprising 3′ exonuclease activity.

Where the component comprising 3′ exonuclease activity is contacted withthe sample contemporaneously with the component required for theextension of the nucleic acid domain, e.g. wherein the polymerase enzymecomprises 3′ exonuclease activity, the extension and degradationreactions will occur simultaneously.

Where the component comprising 3′ exonuclease activity is contacted withthe sample before the component required for the extension of thenucleic acid domain, e.g. wherein an independent enzyme comprising 3′exonuclease activity (and no substantial or detectable polymeraseactivity) is contacted with the sample, the nucleic acid domains withfree and unprotected 3′ ends of unbound probes will be degraded and thecomponent comprising 3′ exonuclease activity may be inactivated (e.g.removed, inhibited or denatured) prior to the extension reactions.However, the 3′ exonuclease activity may be retained during theextension step.

In one embodiment, the component comprising 3′ exonuclease activity isinactivated prior to the step of amplifying the extension product toprevent degradation of components required for the amplification step.The term “inactivated” means that the component is inhibited, denatured,e.g. by heat, or physically removed from the sample. In this respect,only the 3′ exonuclease activity need be inactivated.

For example, wherein the extension product is amplified by PCR, standardunmodified primers (with free and unprotected 3′ ends) would be asubstrate for the 3′ exonuclease and failure to inactivate this activitywould result in degradation of these PCR reagents and therefore limitedor no amplification.

In a preferred embodiment, some or all of the reagents for theamplification reaction are added to the sample before it is contactedwith the component comprising 3′ exonuclease activity. In a particularlypreferred embodiment said reagents are added between steps (b) and (c)as defined above. Alternatively, some or all of the reagents for theamplification reaction are added to the sample at the same time as, i.e.simultaneously or contemporaneously with, the component comprising 3′exonuclease activity.

In one embodiment, the primers are provided in a modified form such thatthey are resistant to 3′ exonuclease activity. Modifications to nucleicacid molecules or to nucleotides contained within a nucleic acidmolecule, to prevent degradation by exonucleases are well known in theart and generally utilise the modification of one or more residues atthe protected end, e.g. the 3′ end. Any modification that is suitablefor the protection against 3′ exonuclease activity may be utilised inthe methods of the invention. In this respect, the primers for use inthe present invention preferably comprise at least one modifiednucleotide at the 3′ end. For instance, the modifications may beselected from any one or more of the list comprising athiophosphate-modified nucleotide, a locked nucleic acid nucleotide(inaccessible RNA nucleotide), a 2′-OMe-CE Phosphoramidite modifiednucleotide, or a peptide nucleic acid nucleotide.

For the step of amplifying the extension product of the method of theinvention it is advantageous to use “hot start” primers (described inKaboev et al., 2000, Nuc. Acids Res., 28(21), pp. e94, which isincorporated herein by reference). Hot start primers are oligonucleotideprimers which comprise a stem-loop structure by virtue of complementaryregions at the 5′ and 3′ ends. In this respect, the primer is designedto be complementary to the target sequence (a specific region in theextension product) and at least 5-6 nucleotides are added to the 5′ endof the primer that form a sequence that is complementary to the 3′ endof the primer. At low temperatures, e.g. the temperature at which thecomponents of the reaction are mixed and/or the extension reaction isperformed, the primers form a stem loop structure and cannot serve as aneffective primer for amplification of the extension product. However,after heating to the annealing temperature of the PCR, the primersacquire a linear structure and primer extension (amplification) canbegin. The use of hot start primers is believed to prevent inference ofthe PCR primers with the interaction between proximity probes of themethod and to further protect said primers from 3′ exonuclease activity.

The term “amplifying” or “amplified” is used generally herein to includeany means of increasing the number of copies of the extension product,or part thereof, in the assay as a means of signalling the presence ofthe target analyte in the sample. For instance, any amplification meansknown in the art may be utilised in the methods of the invention, e.g.PCR, LCR, RCA, MDA etc. Depending on the abundance of the target analytein the sample, it may be necessary to amplify the extension product, orpart thereof, such that the concentration of the extension product hasdoubled, i.e. 2 times the number of copies present before amplification.Alternatively, it may be preferable to increase the number of copies bymultiple orders of magnitude. In some embodiments amplification resultsin the sample comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50or 75 times the original amount of extension product or part thereof. Infurther preferred embodiments it may be preferable to amplify theextension product such that the sample comprises at least 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ etc. times the original amount ofextension product or part thereof.

It will be apparent that it is not necessary to amplify all of anextension product in order to determine whether the sample comprises thetarget analyte. It is necessary to amplify only a portion of theextension product that was not present in the sample before theextension reaction occurred. For example, the extension product willeffectively comprise two parts: a first “old” part (the existing part)containing the nucleotide sequence that made up the nucleic acid domainof the proximity probe (or splint) and a second “new” part (the extendedpart) containing the nucleotide sequence generated by the templatedextension reaction. It is the detection of the second “new” or“extended” part that allows the detection of the of the target analyte,i.e. if there is no analyte, there will be no extension and hence no“new” or “extended” part. Thus, in a preferred aspect of the invention,the step of amplifying the extension product comprises amplifying aportion of the extended part of the extension product.

A portion of the extended part need be of sufficient size that it can bedistinguished from other sequences present in the sample. In effect, theportion of the extended part of the extension product acts as a uniqueidentifier or signal that corresponds to the presence of the targetanalyte. Hence, if the portion comprises a nucleotide sequence that isnot otherwise present in the sample, the amplification of this sequenceis sufficient to signal the presence and quantity of the target analytein the sample.

Thus, the portion may comprise at least 8 nucleotides, preferably atleast 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides.Portions of the extended part of the extension product may generally bein the range of between about 8 up to about 1000 nucleotides in length,where in certain embodiments they may range from about 8 to about 500nucleotides in length including from about 8 to about 250 nucleotides inlength, e.g., from about 8 to about 160 nucleotides in length, such asfrom about 12 to about 150 nucleotides in length, from about 14 to about130 nucleotides in length, from about 16 to about 110 nucleotides inlength, from about 8 to about 90 nucleotides in length, from about 12 toabout 80 nucleotides in length, from about 14 to about 75 nucleotides inlength, from about 16 to about 70 nucleotides in length, from about 16to about 60 nucleotides in length, and so on. In certain representativeembodiments, the extended part of the extension product may range inlength from about 10 to about 80 nucleotides in length, from about 12 toabout 75 nucleotides in length, from about 14 to about 70 nucleotides inlength, from about 34 to about 60 nucleotides in length, and any lengthbetween the stated ranges.

Whilst it is envisaged that the whole of the extension product may beamplified, i.e. both the existing and extended parts, it is sufficientthat the amplification product comprises at least a portion of theextended part of the extension product. In one aspect of the invention,primers may be designed to flank either side of the portion of theextended part of the extension product and amplification of thatportion, e.g. by PCR, and the amplification product (comprising theportion of the extended product) may be detected as described below. Inanother aspect of the invention, the portion of the extended part of theextension product may form the template for the ligation of, e.g. apadlock probe (as described elsewhere herein) to form a circularoligonucleotide and amplification of that sequence, e.g. by rollingcircle amplification (RCA), would result in an amplification productcomprising multiple copies of the sequence corresponding to the portionof extended part of the extension product. Thus, in this way theextension product, or more particularly a part thereof, may beamplified. Alternatively or additionally, the portion of extended partof the extension product may act as a primer for RCA of a circularoligonucleotide comprising a sequence that is complementary to theextended part of the extension product. As above, the resulting productwould comprise multiple copies of the sequence corresponding to theportion of extended part of the extension product, which could bedetected as described below. Thus in this embodiment also the extensionproduct, or more particularly a part thereof, is amplified.

In embodiments where the “splint” oligonucleotide is extended, saidextended oligonucleotide may be circularised to provide a template foramplification, preferably rolling circle amplification. In theseembodiments, the 3′ end (the extended end) of the extendedoligonucleotide is ligated to the 5′ end of the extended oligonucleotide(the non-extended end), wherein said ligation can be mediated by anysuitable means, as described elsewhere herein. In a particularlypreferred embodiment the ligation reaction is a templated ligation,wherein the 3′ and 5′ ends of the extended oligonucleotide are broughtinto proximity to each other by hybridisation to a nucleic acidmolecule, e.g. an oligonucleotide that is added to the reaction to actas a “splint” or “molecular bridge” between the 3′ and 5′ ends of theextended oligonucleotide (as defined elsewhere herein). On hybridisationof the ends of the extended oligonucleotide to the “splint” nucleic acidmolecule, the ends may be ligated, e.g. by the activity of a ligaseenzyme, to form a circular oligonucleotide which contains the extendedpart of the extension product. Hence, amplification of said circularoligonucleotide, e.g. by rolling circle amplification, results in theamplification of the extension product. In this case, the amplificationproduct comprises a sequence that is the complement of the extended partof the extension product. Hence, amplification of the circularisedoligonucleotide results in the indirect amplification of the extensionproduct.

It will be apparent from the above, that the extended part of theextension product need comprise only a relatively small number ofnucleotides. Furthermore, the maximum size of the extension product willbe dependent on the size of the nucleic acid domains of the proximityprobes and/or splint oligonucleotide (as defined below) which act as thetemplate for the extension product. The extension product may resultfrom full or partial extension of the nucleic acid domain and/or splintoligonucleotide, i.e. the extension reaction may result in extensionproducts that have been extended to the end of the template nucleic acidor the extension reaction may be using conditions such that theextension product is only a partial complementary strand of the templatenucleic acid.

The term “detecting” or “detected” is used broadly herein to include anymeans of determining the presence of the analyte (i.e. if it is presentor not) or any form of measurement of the analyte. In the method of theinvention the analyte is detected indirectly by amplifying the extensionproduct (which includes amplifying a product based on, or derived from,or generated using, the extension product) and detecting saidamplification product. Hence, detecting the analyte is equivalent todetecting the amplification product as defined above and these terms areused interchangeably herein.

Thus “detecting” may include determining, measuring, assessing orassaying the presence or absence or amount or location of analyte in anyway. Quantitative and qualitative determinations, measurements orassessments are included, including semi-quantitative. Suchdeterminations, measurements or assessments may be relative, for examplewhen two or more different analytes in a sample are being detected, orabsolute. As such, the term “quantifying” when used in the context ofquantifying a target analyte(s) in a sample can refer to absolute or torelative quantification. Absolute quantification may be accomplished byinclusion of known concentration(s) of one or more control analytesand/or referencing the detected level of the target analyte with knowncontrol analytes (e.g., through generation of a standard curve).Alternatively, relative quantification can be accomplished by comparisonof detected levels or amounts between two or more different targetanalytes to provide a relative quantification of each of the two or moredifferent analytes, i.e., relative to each other.

The “analyte” may be any substance (e.g. molecule) or entity it isdesired to detect by the method of the invention. The analyte is the“target” of the assay method of the invention. The analyte mayaccordingly be any biomolecule or chemical compound it may be desired todetect, for example a peptide or protein, or nucleic acid molecule or asmall molecule, including organic and inorganic molecules. The analytemay be a cell or a microorganism, including a virus, or a fragment orproduct thereof. It will be seen therefore that the analyte can be anysubstance or entity for which a specific binding partner (e.g., anaffinity binding partner) can be developed. All that is required is thatthe analyte is capable of simultaneously binding at least two bindingpartners (more particularly, the analyte-binding domains of at least twoproximity probes). Proximity probe-based assays, such as that of thepresent invention, have found particular utility in the detection ofproteins or polypeptides. Analytes of particular interest may thusinclude proteinaceous molecules such as peptides, polypeptides, proteinsor prions or any molecule which includes a protein or polypeptidecomponent, etc., or fragments thereof. In a particularly preferredembodiment of the invention, the analyte is a wholly or partiallyproteinaceous molecule. The analyte may be a single molecule or acomplex that contains two or more molecular subunits, which may or maynot be covalently bound to one another, and which may be the same ordifferent. Thus in addition to cells or microrganisms, such a complexanalyte may also be a protein complex. Such a complex may thus be ahomo- or hetero-multimer. Aggregates of molecules e.g. proteins may alsobe target analytes, for example aggregates of the same protein ordifferent proteins. The analyte may also be a complex between proteinsor peptides and nucleic acid molecules such as DNA or RNA. Of particularinterest may be the interactions between proteins and nucleic acids,e.g. regulatory factors, such as transcription factors, and DNA or RNA.

All biological and clinical samples are included, e.g. any cell ortissue sample of an organism, or any body fluid or preparation derivedtherefrom, as well as samples such as cell cultures, cell preparations,cell lysates etc. Environmental samples, e.g. soil and water samples orfood samples are also included. The samples may be freshly prepared orthey may be prior-treated in any convenient way e.g. for storage.

Representative samples thus include any material which may contain abiomolecule, or any other desired or target analyte, including forexample foods and allied products, clinical and environmental samples.The sample may be a biological sample, which may contain any viral orcellular material, including all prokaryotic or eukaryotic cells,viruses, bacteriophages, mycoplasmas, protoplasts and organelles. Suchbiological material may thus comprise all types of mammalian andnon-mammalian animal cells, plant cells, algae including blue-greenalgae, fungi, bacteria, protozoa etc. Representative samples thusinclude whole blood and blood-derived products such as plasma, serum andbuffy coat, blood cells, urine, faeces, cerebrospinal fluid or any otherbody fluids (e.g. respiratory secretions, saliva, milk, etc), tissues,biopsies, cell cultures, cell suspensions, conditioned media or othersamples of cell culture constituents, etc. The sample may be pre-treatedin any convenient or desired way to prepare for use in the method of theinvention, for example by cell lysis or purification, isolation of theanalyte, etc.

The proximity probes for use in the method of the invention comprise ananalyte-binding domain and a nucleic acid domain, and are in effectdetection probes which bind to the analyte (via the analyte-bindingdomain), the binding of which may be detected (to detect the analyte) bymeans of detecting the interaction which occurs between the nucleic aciddomains thereof upon such binding. Accordingly the probes may be viewedas nucleic acid-tagged affinity ligands or binding partners for theanalyte, the analyte-binding domain being the affinity binding partner,and the nucleic acid domain the nucleic acid tag. The nucleic aciddomain is coupled to the analyte-binding domain and this “coupling” orconnection may be by any means known in the art, and which may bedesired or convenient and may be direct or indirect e.g. via a linkinggroup. For example, the domains may be associated with one another bycovalent linkage (e.g. chemical cross-linking) or by non-covalentassociation e.g., via streptavidin-biotin based coupling (biotin beingprovided on one domain and streptavidin on the other).

The analyte binding domain may be any binding partner for the targetanalyte, and it may be a direct or indirect binding partner therefor.Thus it may bind to the target analyte directly, or indirectly via anintermediary molecule or binding partner which binds to the targetanalyte, the analyte binding domain binding to said intermediarymolecule (binding partner). Particularly, the analyte-binding domain orthe intermediary binding partner is a specific binding partner for theanalyte. A binding partner is any molecule or entity capable of bindingto its target, e.g. target analyte, and a specific binding partner isone which is capable of binding specifically to its target (e.g. thetarget analyte), namely that the binding partner binds to the target(e.g. analyte) with greater affinity and/or specificity than to othercomponents in the sample. Thus binding to the target analyte may bedistinguished from non-target analytes; the specific binding partnereither does not bind to non-target analytes or does so negligibly ornon-detectably or any such non-specific binding, if it occurs, may bedistinguished. The binding between the target analyte and its bindingpartner is typically non-covalent.

The analyte binding domain may be selected to have a high bindingaffinity for a target analyte. By high binding affinity is meant abinding affinity of at least about 10⁻⁴ M, usually at least about 10⁻⁶ Mor higher, e.g., 10⁻⁹ M or higher. The analyte binding domain may be anyof a variety of different types of molecules, so long as it exhibits therequisite binding affinity for the target protein when present as partof the proximity probe. In other embodiments, the analyte binding domainmay be a ligand that has medium or even low affinity for its targetanalyte, e.g., less than about 10⁻⁴ M.

The analyte binding domain may be a small molecule or large moleculeligand. By small molecule ligand is meant a ligand ranging in size fromabout 50 to about 10,000 daltons, usually from about 50 to about 5,000daltons and more usually from about 100 to about 1000 daltons. By largemolecule is meant a ligand ranging in size from about 10,000 daltons orgreater in molecular weight.

The small molecule may be any molecule, as well as a binding portion orfragment thereof, that is capable of binding with the requisite affinityto the target analyte. Generally, the small molecule is a small organicmolecule that is capable of binding to the target analyte of interest.The small molecule will include one or more functional groups necessaryfor structural interaction with the target analyte, e.g. groupsnecessary for hydrophobic, hydrophilic, electrostatic or even covalentinteractions. Where the target analyte is a protein, the small moleculeligand will include functional groups necessary for structuralinteraction with proteins, such as hydrogen bonding,hydrophobic-hydrophobic interactions, electrostatic interactions, etc.,and will typically include at least an amine, amide, sulfhydryl,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups. The small molecule may also comprise aregion that may be modified and/or participate in covalent linkage tothe nucleic acid domain of the proximity probe, without substantiallyadversely affecting the small molecule's ability to bind to its targetanalyte.

Small molecule affinity ligands often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Also ofinterest as small molecules are structures found among biomolecules,including peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.Such compounds may be screened to identify those of interest, where avariety of different screening protocols are known in the art.

The small molecule may be derived from a naturally occurring orsynthetic compound that may be obtained from a wide variety of sources,including libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including the preparationof randomized oligonucleotides and oligopeptides. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural or synthetically produced libraries and compounds are readilymodified through conventional chemical, physical and biochemical means,and may be used to produce combinatorial libraries. Known smallmolecules may be subjected to directed or random chemical modifications,such as acylation, alkylation, esterification, amidification, etc, toproduce structural analogs.

As such, the small molecule may be obtained from a library of naturallyoccurring or synthetic molecules, including a library of compoundsproduced through combinatorial means, i.e. a compound diversitycombinatorial library. When obtained from such libraries, the smallmolecule employed will have demonstrated some desirable affinity for theprotein target in a convenient binding affinity assay. Combinatoriallibraries, as well as methods for their production and screening, areknown in the art and described in: U.S. Pat. No. 5,741,713; 5,734,018;5,731,423; 5,721,099; 5,708,153; 5,698,673; 5,688,997; 5,688,696;5,684,711; 5,641,862; 5,639,603; 5,593,853; 5,574,656; 5,571,698;5,565,324; 5,549,974; 5,545,568; 5,541,061; 5,525,735; 5,463,564;5,440,016; 5,438,119; 5,223,409, the disclosures of which are hereinincorporated by reference.

The analyte binding domain may also be a large molecule. Of particularinterest as large molecule analyte binding domains are antibodies, aswell as binding fragments and derivatives or mimetics thereof. Whereantibodies are the analyte binding domain, they may be derived frompolyclonal compositions, such that a heterogeneous population ofantibodies differing by specificity are each “tagged” with the same tagnucleic acid (nucleic acid domain) or monoclonal compositions, in whicha homogeneous population of identical antibodies that have the samespecificity for the target analyte are each tagged with the same tagnucleic acid. As such, the analyte binding domain may be either amonoclonal or polyclonal antibody. In yet other embodiments, theaffinity ligand is an antibody binding fragment or derivative or mimeticthereof, where these fragments, derivatives and mimetics have therequisite binding affinity for the target analyte. For example, antibodyfragments, such as Fv, F(ab)₂ and Fab may be prepared by cleavage of theintact protein, e.g. by protease or chemical cleavage. Also of interestare recombinantly or synthetically produced antibody fragments orderivatives, such as single chain antibodies or scFvs, or other antibodyderivatives such as chimeric antibodies or CDR-grafted antibodies, wheresuch recombinantly or synthetically produced antibody fragments retainthe binding characteristics of the above antibodies. Such antibodyfragments or derivatives generally include at least the V_(H) and V_(L)domains of the subject antibodies, so as to retain the bindingcharacteristics of the subject antibodies. Such antibody fragments,derivatives or mimetics of the subject invention may be readily preparedusing any convenient methodology, such as the methodology disclosed inU.S. Pat. Nos. 5,851,829 and 5,965,371; the disclosures of which areherein incorporated by reference.

The above described antibodies, fragments, derivatives and mimeticsthereof may be obtained from commercial sources and/or prepared usingany convenient technology, where methods of producing polyclonalantibodies, monoclonal antibodies, fragments, derivatives and mimeticsthereof, including recombinant derivatives thereof, are known to thoseof the skill in the art.

Also suitable for use as binding domains are polynucleic acid aptamers.Polynucleic acid aptamers may be RNA oligonucleotides which may act toselectively bind proteins, much in the same manner as a receptor orantibody (Conrad et al., Methods Enzymol. (1996), 267(CombinatorialChemistry), 336-367). In certain embodiments where the analyte bindingdomain is a nucleic acid, e.g., an aptamer, the target analyte is not anucleic acid.

Importantly, the analyte binding domain will be one that includes amoiety that can be covalently attached to the nucleic acid domainwithout substantially abolishing the binding affinity of the analytebinding domain to its target analyte.

In addition to antibody-based peptide/polypeptide or protein-basedbinding domains, the analyte binding domain may also be a lectin, asoluble cell-surface receptor or derivative thereof, an affibody or anycorn binatorially derived protein or peptide from phage display orribosome display or any type of combinatorial peptide or proteinlibrary. Combinations of any analyte-binding domain may be used.

The binding sites on the analyte for the respective analyte-bindingdomains of the proximity probes in a set may be the same or different.Thus, for example in the case of a homomeric protein complex oraggregate comprising two or more identical subunits or proteinconstituents, the analyte-binding domains of two or more probes may bethe same. Where the analyte is a single molecule or comprises differentsub-units or constituents (e.g. a heteromeric complex or an aggregate ofdifferent proteins), the analyte binding domains will be different.

Since the length of the nucleic acid domain of the proximity probes canbe constructed to span varying molecular distances, binding sites on theanalyte for the analyte binding domain need not be on the same molecule.They may be on separate, but closely positioned, molecules. For example,the multiple binding domains of an organism, such as a bacteria or cell,or a virus, can be targeted by the methods of the present invention.

As noted above, the analyte-binding domain may bind to the analytedirectly or indirectly. In the case of indirect binding, the targetanalyte may first be bound by a specific binding partner (or affinityligand), and the analyte-binding domain of the proximity probe may bindto the specific binding partner. This enables the design of proximityprobes as universal reagents. For example the analyte-specific bindingpartner may be an antibody, and a universal proximity probe set may beused to detect different analytes by binding to the Fc regions of thevarious different analyte-specific antibodies.

The nucleic acid domains of the first and second proximity probes may beregarded as the nucleic acid “tags” which interact to form a detectableproduct, which may be detected to report the detection of the analyte.The nucleic acid domains may thus be regarded as reactive nucleic acidfunctionalities which interact to provide the signal by means of whichthe analyte is detected (more particularly to form a signal-givingproduct). Put another way, the nucleic acid domains may be regarded as“detection tags”, which interact to form, or enable the formation of, a“detectable” tag or product. When two or more analytes are present inthe same sample they may be detected simultaneously using two or moresets of proximity probes, each set of proximity probes being designed toform on interaction one or more unique nucleic acid sequence extensionproducts or “detectable tags”. These unique “detectable tags” may bedetected and quantified contemporaneously with or after amplification,separately using methods well known in the literature including liquidchromatography, electrophoresis, mass spectrometry, DNA sequencing, DNAarray technology both bead based and planar, and also multi-colourreal-time PCR.

In a preferred embodiment, the detectable tag (i.e. the extensionproduct) is amplified and detected by quantitative PCR (qPCR), which isalso known as real-time PCR. In a particularly preferred embodiment, theqPCR uses a dye which intercalates with nucleic acid molecules toprovide a detectable signal, preferably a fluorescent signal.Fluorescent intercalating dyes that may find particular use in thepresent invention are SYBR Green® and EvaGreen™, although the qPCRembodiments of the invention are not limited to these dyes.

In the method of the present invention, one or both of the nucleic aciddomains of the first and second proximity probes may be extended, whichresults in the formation of a new nucleic acid molecule or “extensionproduct” which may be amplified and detected.

In some embodiments the nucleic acid domains may be ligated together,following the extension of one of the nucleic acid domains, to producethe extension products or detectable tags, i.e. the gap between thenucleic acid domains is “filled in” by a polymerase enzyme using the“splint” oligonucleotide as a template. In embodiments where the nucleicacid domains are ligated, this ligation is mediated by a splint, whichas discussed above, may be considered to be part of the nucleic aciddomain of one of the proximity probes, i.e. wherein the nucleic aciddomain is partially double stranded. The splint may be providedseparately, either as a free nucleic acid molecule or it can be providedas the nucleic acid domain of a third proximity probe.

The ligation results in the formation of a new nucleic acid molecule orsequence, which may be amplified and detected.

The nucleic acid domains may be a single stranded nucleic acid molecule(e.g. an oligonucleotide), a partially double stranded and partiallysingle stranded molecule, or a double stranded molecule that includes aregion that is double stranded and a region where the two nucleic acidstrands are not complementary and therefore single stranded. As such, incertain embodiments, the nucleic acid domain is made up of a singlestranded nucleic acid. In other embodiments, the nucleic acid domain maybe made up of two partially complementary nucleic acid strands, wherethe two strands include a hybridized region and non-hybridized region.

The nucleic acid domains of the first and second proximity probes mustbe capable of interaction by hybridisation, i.e. the formation of one ormore duplexes. This interaction may be direct, e.g. the nucleic aciddomains comprise regions of complementarity to each other, preferably attheir 3′ ends (although the region of complementarity may be internal toone nucleic acid domain, see e.g. Versions 2 and 4 of FIG. 1), orindirect, e.g. the nucleic acid domains of said first and secondproximity probes may each hybridise with a region of a so-called“splint” oligonucleotide.

The nucleic acid domains are generally of a length sufficient to allowinteraction with the nucleic acid domain of another proximity probe whenbound to a target analyte (or splint-mediated interaction). Nucleic aciddomains are usually in the range of between about 8 up to about 1000nucleotides in length, where in certain embodiments they may range fromabout 8 to about 500 nucleotides in length including from about 8 toabout 250 nucleotides in length, e.g., from about 8 to about 160nucleotides in length, such as from about 12 to about 150 nucleotides inlength, from about 14 to about 130 nucleotides in length, from about 16to about 110 nucleotides in length, from about 8 to about 90 nucleotidesin length, from about 12 to about 80 nucleotides in length, from about14 to about 75 nucleotides in length, from about 16 to about 70nucleotides in length, from about 16 to about 60 nucleotides in length,and so on. In certain representative embodiments, the nucleic aciddomain may range in length from about 10 to about 80 nucleotides inlength, from about 12 to about 75 nucleotides in length, from about 14to about 70 nucleotides in length, from about 34 to about 60 nucleotidesin length, and any length between the stated ranges. In someembodiments, the nucleic acid domains are usually not more than about28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 46, 50,55, 60, 65, or 70 nucleotides in length.

The nucleic acid domain may be made up of ribonucleotides and/ordeoxyribonucleotides as well as synthetic nucleotide residues that arecapable of participating in Watson-Crick type or analogous base pairinteractions, i.e. “hybridisation” or the formation of a “duplex”. Thus,the nucleic acid domain may be DNA or RNA or any modification thereofe.g. PNA or other derivatives containing non-nucleotide backbones.

The sequence of the nucleic acid domain of the first and secondproximity probes (i.e. the “detection” nucleic acid domains) may be anysequences which are capable of forming a duplex and may be chosen orselected with respect to each other or the splint, if present. Thus, thesequence is not critical as long as the first and second domains mayhybridise to each other or a third nucleic acid domain (splint).However, the sequences should be chosen to avoid the occurrence ofhybridization events other than between the nucleic acid domains of thefirst and second proximity probes or with that of the splintoligonucleotide. Once the sequence is selected or identified, thenucleic acid domains may be synthesized using any convenient method.

The two components of the proximity probe are joined together eitherdirectly through a bond or indirectly through a linking group. Wherelinking groups are employed, such groups may be chosen to provide forcovalent attachment of the nucleic acid and analyte-binding domainsthrough the linking group, as well as maintain the desired bindingaffinity of the analyte-binding domain for its target analyte. Linkinggroups of interest may vary widely depending on the analyte-bindingdomain. The linking group, when present, is in many embodimentsbiologically inert. A variety of linking groups are known to those ofskill in the art and find use in the subject proximity probes. Inrepresentative embodiments, the linking group is generally at leastabout 50 daltons, usually at least about 100 daltons and may be as largeas 1000 daltons or larger, for example up to 1000000 daltons if thelinking group contains a spacer, but generally will not exceed about 500daltons and usually will not exceed about 300 daltons. Generally, suchlinkers will comprise a spacer group terminated at either end with areactive functionality capable of covalently bonding to the nucleic acidor analyte binding moieties. Spacer groups of interest may includealiphatic and unsaturated hydrocarbon chains, spacers containingheteroatoms such as oxygen (ethers such as polyethylene glycol) ornitrogen (polyamines), peptides, carbohydrates, cyclic or acyclicsystems that may possibly contain heteroatoms. Spacer groups may also becomprised of ligands that bind to metals such that the presence of ametal ion coordinates two or more ligands to form a complex. Specificspacer elements include: 1,4-diaminohexane, xylylenediamine,terephthalic acid, 3,6-dioxaoctanedioic acid,ethylenediamine-N,N-diacetic acid,1,1′-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid),4,4′-ethylenedipiperidine. Potential reactive functionalities includenucleophilic functional groups (amines, alcohols, thiols, hydrazides),electrophilic functional groups (aldehydes, esters, vinyl ketones,epoxides, isocyanates, maleimides), functional groups capable ofcycloaddition reactions, forming disulfide bonds, or binding to metals.Specific examples include primary and secondary amines, hydroxamicacids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates,oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters,glycidyl ethers, vinylsulfones, and maleimides. Specific linker groupsthat may find use in the subject proximity probes includeheterofunctional compounds, such as azidobenzoyl hydrazide,N[4-(p-azidosalicylamino)butyl]-3′-[2′-pyridyldithio]propionamid),bis-sulfosuccinimidyl suberate, dimethyladipimidate,disuccinimidyltartrate, N-maleimidobutyryloxysuccinimide ester,N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl[4-azidophenyl]-1,3′-dithiopropionate, N-succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, andsuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate,3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP),4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimideester (SMCC), and the like.

The proximity probes employed in the subject methods may be preparedusing any convenient method. In representative embodiments, nucleic aciddomains may be conjugated to the analyte-binding domain, either directlyor through a linking group. The components can be covalently bonded toone another through functional groups, as is known in the art, wheresuch functional groups may be present on the components or introducedonto the components using one or more steps, e.g. oxidation reactions,reduction reactions, cleavage reactions and the like. Functional groupsthat may be used in covalently bonding the components together toproduce the proximity probe include: hydroxy, sulfhydryl, amino, and thelike. The particular portion of the different components that aremodified to provide for covalent linkage may be chosen so as not tosubstantially adversely interfere with that component's desired bindingaffinity for the target analyte. Where necessary and/or desired, certainmoieties on the components may be protected using blocking groups, as isknown in the art, see e.g. Green & Wuts, Protective Groups in OrganicSynthesis (John Wiley & Sons) (1991). Methods for producing nucleicacid/antibody conjugates are well known to those of skill in the art.See e.g. U.S. Pat. No. 5,733,523, the disclosure of which is hereinincorporated by reference.

In other embodiments, proximity probes may be produced using in vitroprotocols that yield nucleic acid-protein conjugates, i.e. moleculeshaving nucleic acids, e.g. coding sequences, covalently bonded to aprotein, i.e. where the analyte-binding domain is produced in vitro fromvectors which encode the proximity probe. Examples of such in vitroprotocols of interest include: RepA based protocols (see e.g.,Fitzgerald, Drug Discov. Today (2000) 5:253-258 and WO 98/37186),ribosome display based protocols (see e.g., Hanes et al., Proc. NatlAcad. Sci. USA (1997) 94:4937-42; Roberts, Curr Opin Chem Biol (1999)June; 3: 268-73; Schaffitzel et al., J Immunol Methods (1999) December10; 231: 119-35; and WO 98/54312), etc.

In embodiments which utilise a splint oligonucleotide (which may be anucleic acid domain of a third proximity probe), said splintoligonucleotide functions to mediate the interaction between the nucleicacid domains of the first and second proximity probes (i.e. the“detection” domains). As noted above, the splint oligonucleotide mayalso act as a nucleic acid domain to be extended, resulting in anextension product to be amplified and detected in accordance with themethod of the invention. Thus, the splint may accordingly be viewed a“connector” oligonucleotide which acts to connect or “hold together” thedetection domains of the first and second proximity probes, such theymay interact, or may be ligated together. Alternatively or additionally,the splint may be viewed as the extendible domain of a nucleic aciddomain or tag or a separate nucleic acid domain which acts as a “primer”for extension to generate the extension product for amplification anddetection.

In these embodiments the splint hybridises with the nucleic acid domainsof the first and second proximity probes. More particularly, the splinthybridises (anneals) simultaneously with the nucleic acid domains of atleast the first and second proximity probes. However, wherein the“splint” oligonucleotide is prehybridised to the nucleic acid domain ofat least one of said proximity probes, it will be hybridised with thenucleic acid domain of one proximity probe before it hybridises with theother. Nevertheless, the “splint” will preferably hybridise with thenucleic acid domains of both proximity probes at the same time to enablethe formation of a stable complex capable of being extended.

When the splint oligonucleotide is provided as the nucleic acid domainof a third proximity probe, this hybridisation of the nucleic aciddomains of all of the set of proximity probes to each other increasesthe avidity of the probe-target complex upon binding to the targetanalyte. This avidity effect contributes to the sensitivity of the assayby supporting the formation of signal-giving proximity probe-targetanalyte complexes.

The term “hybridisation” or “hybridises” as used herein refers to theformation of a duplex between nucleotide sequences which aresufficiently complementary to form duplexes via Watson-Crick basepairing. Two nucleotide sequences are “complementary” to one anotherwhen those molecules share base pair organization homology.“Complementary” nucleotide sequences will combine with specificity toform a stable duplex under appropriate hybridization conditions. Forinstance, two sequences are complementary when a section of a firstsequence can bind to a section of a second sequence in an anti-parallelsense wherein the 3′-end of each sequence binds to the 5′-end of theother sequence and each A, T(U), G and C of one sequence is then alignedwith a T(U), A, C and G, respectively, of the other sequence. RNAsequences can also include complementary A=U or U=A base pairs. Thus,two sequences need not have perfect homology to be “complementary” underthe invention. Usually two sequences are sufficiently complementary whenat least about 85% (preferably at least about 90%, and most preferablyat least about 95%) of the nucleotides share base pair organization overa defined length of the molecule or the domains that are determined tobe complementary. The nucleic acid domains of the first and secondproximity probes thus contain a region of complementarity for thenucleic acid domain the other proximity probe. Alternatively, where asplint oligonucleotide is used, the first and second proximity probescontain a region of complementarity for the splint oligonucleotide(which may be present on a third proximity probe), and conversely thesplint oligonucleotide contains regions of complementarity for each ofthe nucleic acid domains of the first and second proximity probes.

The regions of complementarity (i.e. hybridisation regions) may have alength in the range of 4-30 bp e.g. 6-20, 6-18, 7-15 or 8-12 bp.

The splint nucleic acid domain is generally of a length sufficient toprovide for the above described simultaneous binding of nucleic aciddomains of the first and second probes. In representative embodiments,the splint oligonucleotides range in length from about 6 to about 500nucleotides, including from about 20 to about 40 nucleotides, e.g. fromabout 25 to about 30 nucleotides.

As noted above, the interaction between the nucleic acid domains of thefirst and second proximity probes is primarily the formation of aduplex, wherein one or both of the nucleic acid domains may be extended,particularly template-directed extension using the nucleic acid domainof the other proximity probe as the template. This may result in theformation of a completely double stranded nucleic acid, e.g. where bothdomains are extended fully, or a partially double stranded molecule,e.g. where only one strand is extended or both strands are extendedpartially. Thus extension of one or both of the nucleic acid domains maybe considered to be a “joining” of the respective domains, e.g. theproduction of one double stranded nucleic acid from two single strandedmolecules.

In other embodiments, this “joining” may be a ligation, particularlywherein, e.g. the nucleic acid domain of the first proximity probe isextended such that its 3′ end is in proximity with the 5′ end of thesecond proximity probe to allow the template-directed ligation of thetwo domains. In such a case, it will clearly be understood that theligation template will be provided by the splint, which may form part ofone of the nucleic acid domains or may be provided separately free insolution or as a nucleic acid domain of a third proximity probe. Such aligation may be carried out using a ligase enzyme, which may, e.g. beadded to the reaction after the polymerase mediated extension of thenucleic acid domain of the first proximity probe.

Thus, in a preferred embodiment of the method of the invention, thenucleic acid domains of the first and second probes are ligatable bymeans of a reaction templated by the hybridised splint, said nucleicacid domains are ligated (following extension of one of the nucleic aciddomains) and the “ligation” product, which is also an extension product,is amplified and detected. In such an embodiment, the splint maytherefore be viewed as a “splint template” or “ligation template” or“template oligonucleotide”. In such embodiments, the splint may also beextended to produce a further “extension product”, which amplified anddetected.

As discussed above, for the various interactions between the nucleicacid domains to take place, the nucleic acid domains of the first andsecond proximity probes need to be coupled to the analyte-bindingdomains in certain orientations. For example, for the extension of bothdomains wherein said domains comprise single stranded nucleic acids,each nucleic acid domain must be coupled to the analyte-binding domainby its 5′ end, leaving a free 3′ hydroxyl end, which may “anneal” or“hybridise” when in proximity. However, for the extension of a singledomain and/or the conjugation of the two domains, it is typical(although not essential, see Version 4 of FIG. 1) to couple the nucleicacid domain of a first proximity probe by it 5′ end (leave a free 3′hydroxyl end, which may be extended) while the other domain will becoupled via its 3′ end (leaving a free 5′ phosphate end, which cannot beextended using a convention polymerase).

In embodiments wherein the nucleic acid domains are ligatable, therespective first and second nucleic acid domains hybridise to the splintor wherein the nucleic acid domain of one of the proximity probes ispartially double stranded with a singled stranded overhang, the nucleicacid domain of the other proximity probe hybridises with a domain of theoverhang. The domain with the 3′ end may then be extended by templatedirected extension up to the 5′ phosphate of the other domain, wherein aligase enzyme may be utilised to join the two strands together. Thus,the respective 3′ and 5′ ends are not be hybridised immediately adjacentto one another on the splint (template) but hybridise to the splintleaving a space (or a stretch of nucleotides) between them.

The gap or space or stretch of nucleotides between the two ends is inthe range of between about 8 up to about 1000 nucleotides in length,where in certain embodiments they may range from about 8 to about 500nucleotides in length including from about 8 to about 250 nucleotides inlength, e.g., from about 8 to about 160 nucleotides in length, such asfrom about 12 to about 150 nucleotides in length, from about 14 to about130 nucleotides in length, from about 16 to about 110 nucleotides inlength, from about 8 to about 90 nucleotides in length, from about 12 toabout 80 nucleotides in length, from about 14 to about 75 nucleotides inlength, from about 16 to about 70 nucleotides in length, from about 16to about 60 nucleotides in length, and so on. In certain representativeembodiments, the nucleic acid domain may range in length from about 10to about 80 nucleotides in length, from about 12 to about 75 nucleotidesin length, from about 14 to about 70 nucleotides in length, from about34 to about 60 nucleotides in length, and any length between the statedranges. In some embodiments, the nucleic acid domains are usually notmore than about 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 44, 46, 50, 55, 60, 65, or 70 nucleotides in length.

Thus, the splint may include a first 3′ region of complementarity forthe nucleic acid domain of the 5′ free proximity probe and a second 5′region of complementarity for the nucleic acid domain of the 3′ freeproximity probe. The first and second regions of the splint may be 3 to20, 6 to 17, 6 to 15 or 6 to 12 or 8 to 12 nucleotides in length, e.g.about 13 to 17, 12 to 16, 11 to 15, or 12 to 14 nucleotides in length orabout 6 to 12, 7 to 11 or 8 to 10 nucleotides in length.

As will be described in more detail below, amplification of theligation/extension product is performed before or contemporaneously withthe detection process. Accordingly, it may in some embodiments bedesirable to design the splint so as to minimise any false amplificationwhich may take place in such a step, for example any possibility of thesplint acting as a template for the polymerase used in theamplification. Thus for example the splint may be provided as an RNAoligonucleotide or a DNA/RNA hybrid; Taq polymerase typically used inamplification reactions cannot use an RNA template. Alternatively, asimilar effect may be achieved using a DNA splint with two shorthybridisation regions; since the hybridisation is weak, such a splintwill not template DNA polymerisation at the high temperatures used inPCR.

Alternatively, in other preferred embodiments the splint isadvantageously extended to produce an extension product as definedabove, which will itself be amplified and detected in accordance withthe methods of the invention.

In certain embodiments a sample may be assayed for two or more differenttarget analytes. In such embodiments, the sample is contacted with a setof proximity probes for each target analyte, such that the number ofsets contacted with the sample may be two or more, e.g., three or more,four or more etc. Such methods find particular use in multiplex andhigh-throughput applications.

The amount of proximity probes that is added to a sample may be selectedto provide a sufficiently low concentration of proximity probe in thereaction mixture to ensure that the proximity probes will not randomlycome into close proximity with one another in the absence of binding toa target analyte, at least not to any great or substantial degree. Assuch, it is intended that only when the proximity probes bind theanalyte through the binding interaction between the analyte-bindingdomains of the proximity probes and the binding sites of the analyte, dothe proximity probes come into close proximity to one another.

However, it has been surprisingly found that the addition of a componentcomprising 3′ exonuclease activity may reduce the number of interactionsbetween proximity probes that are not bound to the target analyte, bydegrading nucleic acid domains with a free and unprotected 3′ end. Inthis respect, such interactions are less stable than those betweenprobes bound to the target analyte and are therefore transient.Consequently, said unbound probes are subject to degradation by thecomponent comprising 3′ exonuclease activity. Thus, in the methods ofthe present invention it may be possible to use concentrations ofproximity probes that could not be used previously in proximity baseddetection assays. This is particularly advantageous where theanalyte-binding domains of the proximity probes have medium or lowaffinity for the target analyte and are therefore required at higherconcentrations.

In representative embodiments, the concentration of the proximity probesin the reaction mixture following combination with the sample rangesfrom about 1 fM to 1 μM, such as from about 1 pM to about 1 nM,including from about 1 pM to about 100 nM.

Following combination of the sample and set(s) of proximity probes, thereaction mixture may be incubated for a period of time sufficient forthe proximity probes to bind target analyte, if present, in the sample.In representative embodiments, the product mixture may be incubated fora period of time ranging from about 5 minutes to about 48 hours,including from about 30 minutes to about 12 hours, at a temperatureranging from about 4 to about 50° C., including from about 20 to about37° C. Conditions under which the reaction mixture is maintained shouldbe optimized to promote specific binding of the proximity probe to theanalyte, while suppressing unspecific interaction. Conditions shouldalso allow for efficient and specific hybridization between the nucleicacid domains as described above.

In some embodiments, the proximity probes are lyophilized. In one aspectof the invention, said lyophilized proximity probes are rehydrated priorto contact with the sample containing the analyte. In a preferred aspectof the invention, the lyophilized proximity probes are rehydrated uponaddition of the sample comprising the target analyte.

In certain embodiments, the effective volume of the incubation mixtureis reduced, at least during the portion of the incubation step in whichthe proximity probes are binding to target analyte, if present in thesample. In these embodiments, the effective volume of the incubationmixture may be reduced for a number of different reasons. In certainembodiments, the effective volume of the incubation mixture is reducedin order to allow for the use of medium and low affinity analyte-bindingdomains and/or increase the sensitivity of the assay. For example, incertain embodiments where the effective volume of the incubation mixtureis reduced, the analyte-binding domains may be medium or low affinitybinders, by which is meant that the analyte-binding domains may have abinding affinity for their target analyte that is less than about 10⁻⁴M, such as about 1 nM Kd. In certain embodiments, the sensitivity of theassay may be increased such that the assay can detect as few as about100 or fewer target analytes in a 1 μl sample, including as few as about75 or fewer target analytes in a 1 μl sample, including as few as about50 or fewer target analytes in a 1 μl sample.

In certain embodiments, a “crowding agent” or “volume excluder” isincluded in the mixture during the incubation step reviewed above, e.g.,to reduce the effective volume of the incubation mixture during bindingof the proximity probes to their target analyte. Typically, the“crowding agent” is a water soluble macromolecular material. Suitablemacromolecular materials broadly comprise biocompatible natural orsynthetic polymers having an average molecular weight of from about 1500to several million, which do not specifically interact with the otherreagents in the mixture, or the product. Such polymers are known in theart as “volume-excluders”, as their primary function is to occupy volumein the in vitro reaction medium and provide a highly concentratedenvironment for biochemical reactions, e.g., approximating in vivoconditions. The volume-excluding polymers must of course be sufficientlysoluble to provide the required concentration. Suitable exemplarypolymers include, but are not limited to: commercially availablepolyethylene glycol (PEG) polymers, e.g., having an average molecularweight greater than about 2000, FICOLL polymers such as those having anaverage molecular weight of about 70,000, bovine plasma albumin,glycogen, polyvinylpyrrolidone, dextran, e.g. sephadex® which iscross-linked dextran, etc. PEG polymers of higher molecular weights,especially, PEG 1450, PEG 3350, PEG 6000 (also sold as PEG 8000), andPEG 20,000, having average molecular weights of about 1450, 3000-3700,6000-7500, and 15,000-20,000, respectively, are employed inrepresentative embodiments. PEG 6000 and PEG 8000 are employed inrepresentative embodiments. The concentration of the volume-excludingpolymers in the incubation reaction in representative embodiments fallswithin a range of about 5% w/v to about 45% w/v, depending upon the typeof polymer and its molecular weight. In general, it is expected that agiven type of polymer of higher molecular weight need be present inlower concentration than the same type of polymer of lower molecularweight to achieve the same effect on enzyme activity.

In a preferred embodiment, the crowding agent or volume excluder issephadex. In a particularly preferred embodiment, the sephadex is typeG-100.

In those embodiments where a volume excluder is employed, prior to thenext step of the method, the incubation mixture may be diluted toaccount for the presence of the volume excluder, e.g., by at least about2-fold or more, such as at least about 5-fold or more, including atleast about 10-fold or more, depending on the amount of volume excluderthat is present, the nature of the dilution fluid, etc., where inrepresentative embodiments the dilution fluid is water or some othersuitable aqueous fluid of water and one or more solutes, e.g., salts,buffering agents, etc.

Instead of, or in addition to, the use of a volume excluder, theincubation mixture may be reduced in volume during incubation byremoving a portion of the water from the incubation mixture, e.g., viaevaporation. In these embodiments, the volume of the fluid may bereduced by at least about 2-fold or more, such as at least about 5-foldor more, including at least about 10-fold or more, as desired.Importantly, not all of the water is removed from the incubation mixturein these embodiments. Any convenient protocol may be employed forreducing the volume of the incubation mixture by removing a selectportion of the water therefrom. An instrument for controllingevaporation rate by monitoring and adjusting humidity and temperaturemay be employed, where in certain embodiments the volume of theincubation mixture is monitored, e.g., by continuously measuring thevolume of the incubation mixture, where when appropriately evaporated,the polymerase and PCR-mixes may be added, as described above. Asdesired, a heating block could be used to enhance the evaporation.Alternatively, the volume of the incubation mixture may be reduced byfiltrating out water. In representative embodiments, a size exclusionfilter is used to selectively contain molecules of sizes larger than acut off limit while smaller molecules and water is removed by passagethrough the filter. The force placed on the solution to move it throughthe filter may be by either centrifugation or vacuum suction.

Upon binding of the binding domains of the proximity probes to theanalyte, the nucleic acid domains of the proximity probes come intoclose proximity to one another. As a result, the nucleic acid domains ofthe first and second probes are able to hybridise to each other directlyor indirectly, e.g. via a splint.

The splint, where present, may be added to the sample before, at thesame time as, or after the proximity probes. In one embodiment thesplint is pre-hybridised to the proximity probe. In another embodiment,the splint oligonucleotide forms part of the nucleic acid domain of theproximity probe. In yet a further embodiment, the splint oligonucleotidemay be coupled to an analyte-binding domain in the form of a thirdproximity probe, wherein it is preferably added at the same time as thefirst and second proximity probes.

Following the combination of the sample with the proximity probes, thesample may be diluted, preferably by the addition of the non-enzymaticcomponents of the extension reaction, e.g. buffers, salts, nucleotidesetc, but not the component comprising 3′ exonuclease activity. In apreferred embodiment, the dilution also comprises the reagents for theamplification reaction, e.g. buffers, salts, nucleotides, primers andpolymerase. The dilution step acts to reduce the possibility ofinteractions between the unbound proximity probes and/or theirinteraction with other components in the sample.

Dilution may also disrupt the interaction between the nucleic aciddomains of the bound probes. However, as the bound probes are in closeproximity any interactions that are disrupted will stabilise (i.e.re-anneal or re-hybridise) under the appropriate conditions. Thus in oneembodiment the sample may be incubated for a further period of timesufficient for the interaction between the bound proximity probes tostabilise. In representative embodiments, the product mixture may beincubated for a period of time ranging from about 5 minutes to about 48hours, including from about 30 minutes to about 12 hours, at atemperature ranging from about 4 to about 50° C., including from about20 to about 37° C. Conditions under which the reaction mixture isincubated should be optimized to maintain specific binding of theproximity probe to the analyte, while suppressing unspecificinteraction. Conditions should also allow for efficient and specifichybridization between the nucleic acid domains as described above.

Where the amplification reagents are added to the sample following thestep of incubating the probes with the sample it is preferred that theprimers are protected from 3′ exonuclease activity, e.g. by modificationof their 3′ ends as described above. In a further preferred embodimentthe primers may alternatively or additionally be hot start PCR primers,e.g. stem loop primers. In a preferred embodiment, the polymerase is athermostable polymerase as defined further below. In yet anotherpreferred embodiment, the buffer and/or salts allow the activity of allenzymes to be added to the sample.

Further to any dilution steps, the component comprising 3′ exonucleaseactivity is then contacted with the sample. This step may includefurther dilution of the sample, e.g. addition of the component withappropriate buffers and/or other salts/components. As described above,the component comprising 3′ exonuclease activity may be added before orcontemporaneously with the polymerase enzyme required for the extensionreaction. In a preferred embodiment, the polymerase enzyme comprisesalso 3′ exonuclease activity. The sample may be further incubated underthe appropriate conditions to allow the 3′ exonuclease activity to acton the nucleic acid domains of unbound proximity probes. The conditionsshould also be conducive to the extension of the nucleic acid domains,if the polymerase is present in the sample. In some embodiments thepolymerase may be added after the component comprising 3′ exonucleaseactivity, wherein the sample may be further incubated to allow theextension products to be generated. Incubation conditions will depend onthe components used in the reaction and some representative conditionsare described above. However, in embodiments in which the polymeraseused to extend the nucleic acid domains is a hyperthermophilicpolymerase as defined above, e.g. a modified Taq polymerase, Pfu DNApolymerase, Pwo DNA polymerase etc, or a thermostable polymerase such asTaq polymerase, other reaction conditions may be preferred in theexonuclease and/or extension phases of the method, particularlytemperature conditions. For instance, the temperature for the extensionreaction and, if the polymerase also has 3′ exonuclease activity, theexonuclease reaction, may range from about 4 to about 80° C., includingfrom about 20 to about 75° C., from about 30 to about 60° C., or fromabout 45 to about 55° C. Thus, in some embodiments the temperature forthe extension reaction and, if the polymerase also has 3′ exonucleaseactivity, the exonuclease reaction, may be at least 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 60 or 75° C.

Following the generation of extension products the component comprising3′ exonuclease activity may be inactivated. In a preferred embodimentthe 3′ exonuclease activity is inactivated by heat denaturation, e.g.65-80° C. for 10 minutes, although it will be apparent that the requiredconditions will vary depending on the nature of the component, e.g.hyperthermophilic polymerases comprising 3′ exonucleases may not beinactivated by the above conditions. In some embodiments, the heatinactivation may be the first step of the amplification reaction.

Where the amplification reactants were not added to the sample followingthe step of contacting the proximity probes with the sample, saidreactants should be contacted with the sample at this stage. In apreferred embodiment, an aliquot of the sample may be transferred to anew vessel comprising the amplification components, for amplificationand detection. In this respect, following the inactivation of thecomponent comprising 3′ exonuclease activity, the primers need not beresistant to 3′ exonuclease activity.

In some embodiments, the polymerase capable of extending the nucleicacid domains of the proximity probes may also be useful in theamplification reaction, e.g. if the polymerase is a hyperthermophilic orthermostable polymerase as described above, e.g. Pfu DNA polyermase, PwoDNA polymerase etc.

Where the amplification reagents were added to the sample following thestep of contacting the proximity probes with the sample, it is possibleto proceed directly with the amplification reaction. In a preferredembodiment, an aliquot of the reaction mixture is transferred to a newvessel for amplification and detection.

In general, any convenient protocol that is capable of detecting thepresence of proximity dependent interactions may be employed. Thedetection protocol may or may not require a separation step.

In one representative embodiment (other representative embodiments ofthe methods of the invention are described above), the extensionproduct(s) generated from the interaction of the first and secondproximity probes is achieved by nucleic acid extension of the free 3′hydroxyl ends of the nucleic acid domains of the first and secondproximity probes, and this interaction is detected by subsequentamplification and detection of the extension product. In thisrepresentative embodiment, extension of the nucleic acid domains of thefirst and second proximity probes is achieved by contacting the reactionmixture with a nucleic acid extending activity, e.g. provided by asuitable nucleic acid polymerase, and maintaining the mixture underconditions sufficient for extension of the nucleic acid domains tooccur.

As is known in the art, polymerases catalyze the formation of aphosphodiester bond between juxtaposed nucleotides, wherein thenucleotide comprising the 3′ hydroxyl moiety (3′ end) may form part ofan existing polymer of nucleotides, i.e. a nucleic acid. Typically, thenucleic acid is annealed or hybridized to a complementary nucleic acidmolecule which acts to template (i.e. a template nucleic acid) theextension of the nucleic acid with the free 3′ end. A free nucleotidewith a free 5′ phosphate moiety that is complementary to the nextnucleotide on the template nucleic acid is then joined to the nucleicacid with the free 3′ end to extend it and this process is repeated,e.g. until the end of the template is reached. Any convenient polymerasemay be employed, where representative polymerases of interest include,but are not limited to, polymerases comprising also a component with 3′exonuclease activity, e.g. T4 DNA polymerase, T7 DNA polymerase, Phi29(Φ29) DNA polymerase, DNA polymerase I, Klenow fragment of DNApolymerase I, Pfu DNA polymerase and/or Pwo DNA polymerase. Polymeraseswith 3′ exonuclease activity may be obtained from any suitableorganisms, including but not limited to, prokaryotic, eukaryotic, orarchael organisms. Certain RNA polymerases may also be employed in themethods of the invention.

Where the extension step is performed (the extension product isgenerated) by a polymerase without 3′ exonuclease activity, e.g. Klenowexo(−), Taq polymerase, Pfu (exo⁻) DNA polymerase, Pwo (exo⁻) DNApolymerase etc, a component comprising 3′ exonuclease, e.g. anexonuclease enzyme, is added to the sample before, or contemporaneouslywith, the polymerase. Any convenient 3′ exonuclease may be employed,e.g. exonuclease I. Certain RNA exonucleases may also be employed in themethods of the invention.

In this extension step, a suitable polymerase and, if requiredadditional 3′ exonuclease, and any reagents that are necessary and/ordesirable are combined with the reaction mixture and maintained underconditions sufficient for extension of the hybridized nucleic aciddomains to occur. Said conditions should also be sufficient fordegradation of the nucleic acid domains of unbound proximity probes tooccur. Polymerase and exonuclease reaction conditions are well known tothose of skill in the art. During extension and/or degradation, thereaction mixture in certain embodiments may be maintained at atemperature ranging from about 4° C. to about 80° C., such as from about20 to about 75° C., from about 30 to about 60° C., e.g. from about 45 toabout 55° C. or from about 20° C. to about 37° C. (depending on theoptimum conditions for the polymerase used in the assay) for a period oftime ranging from about 5 seconds to about 16 hours, such as from about1 minute to about 1 hour. In yet other embodiments, the reaction mixturemay be maintained at a temperature ranging from about 35° C. to about45° C., such as from about 37° C. to about 42° C., e.g., at or about 38°C., 39° C., 40° C. or 41° C., or ranging from about 35° C. to about 75°C., such as from above 40° C. to about 60° C. such as from about 45° C.to about 55° C., e.g., at or about 46° C., 47° C., 48° C., 49° C., 50°C., 51° C., 52° C., 53° C., or 54° C., for a period of time ranging fromabout 5 seconds to about 16 hours, such as from about 1 minute to about1 hour, including from about 2 minutes to about 8 hours. In arepresentative embodiment wherein the amplification components are notincluded before addition of the component comprising 3′ exonucleaseactivity, the extension and degradation reaction mixture includes 70 mMTris pH7.5, 17 mM ammonium sulfate, 1 mM DTT, 40 μM of each dNTP, 3 mMMgCl₂, 62.5 units/ml DNA polymerase, e.g. T4 DNA polymerase. In arepresentative embodiment wherein the amplification components areincluded before addition of the component comprising 3′ exonucleaseactivity, the extension and degradation reaction mixture includes 50 mMKCl, 20 mM Tris HCl pH8.4, 0.2 mM of each dNTP, 3 mM MgCl₂, SYBR GreenI, 20 nM fluorscein, 3′-thioate protected hairpin primers, 25 units/mliTaq DNA polymerase, and 62.5 units/ml DNA polymerase, e.g. T4 DNApolymerase.

Following extension, the extension products (e.g. extended nucleic aciddomains of the first and second probes) are amplified and detected as anindication of the presence, or as a measure of the amount and optionallythe location, of analyte in the sample. As described above, the extendedproduct may comprise a single stranded or double stranded nucleic acidmolecule. A single stranded nucleic acid molecule may result from theextension of the splint oligonucleotide which may be dissociated fromthe nucleic acids of the first and second proximity probes or theproduct of the conjugation of the two proximal nucleic acid domains ofthe first and second probes terminating at each end in an analytebinding domain.

The next step of the method following the extension step is to determinethe presence of the extended product in the reaction mixture in order todetect the target analyte in the sample. In other words, the reactionmixture is screened etc. (i.e., assayed, assessed, evaluated, tested,etc.) for the presence of any resultant extension products in order todetect the presence of the target analyte in the sample being assayed.According to the present invention the detection step involves anamplification step to generate an amplification product which isdetected, typically amplification of all or a portion of the extensionproduct.

The extension product, or more particularly the amplification product,produced by the above-described methods may, in the broadest sense, bedetected using any convenient protocol. The particular detectionprotocol may vary depending on the sensitivity desired and theapplication in which the method is being practiced. In the method of theinvention as described herein, the detection protocol may include anamplification component, in which the copy number of the extensionproduct nucleic acid (or part thereof) is increased, e.g., to enhancesensitivity of the particular assay. However, it is possible that inother methods the extension product may be directly detected without anyamplification.

Although not a preferred embodiment of the method of the invention,where detection without amplification is practicable, the nucleic acidextension product may be detected in a number of different ways. Forexample, one or more of the extension product may be directly labelled,e.g., fluorescently, or otherwise spectrophotometrically, orradioisotopically labelled or with any signal-giving label, such thatthe extension product is directly labelled. In these embodiments, thedirectly labelled extension product may be size separated from theremainder of the reaction mixture, including unextended oligonucleotides(i.e. nucleic acid domain oligonucleotides or splint oligonucleotides),in order to detect the extended nucleic acid. Alternatively,conformationally selective probes, e.g., molecular beacons (as describedin greater detail below) may be employed to detect to the presence ofthe extension product, where these probes are directed to a sequencethat is only present in the extended nucleic acid product.

As indicated above, in a preferred embodiment of the subject methods,the detection step includes an amplification step, where the copy numberof extended nucleic acid or part thereof is increased, e.g., in order toenhance sensitivity of the assay. The amplification may be linear orexponential, as desired, where representative amplification protocols ofinterest include, but are not limited to: polymerase chain reaction(PCR); isothermal amplification, Rolling circle amplification, etc. In aparticularly preferred embodiment of the invention, the amplificationprotocol is quantitative-PCR (qPCR) or real-time PCR.

Rolling circle amplification using padlock probes, e.g. as described inU.S. Pat. No. 6,558,928, or indeed any circular nucleic acid molecule asa template can also be useful in amplifying an existing “signal” nucleicacid molecule or part thereof, e.g. an extension product generated froma proximity extension assay. Thus, in a preferred aspect of method, theextension product (or part thereof) may be amplified by rolling circleamplification. In one embodiment, RCA is performed using padlock probes.In another embodiment, RCA is performed using circular templates(circular oligonucleotides).

Where the detection step includes an amplification step (morespecifically a step of in vitro amplification of the extension productor part thereof), the amplified product (or amplification product) maybe detected, to detect the analyte.

The polymerase chain reaction (PCR) is well known in the art, beingdescribed in U.S. Pat. Nos.: 4,683,202; 4,683,195; 4,800,159; 4,965,188and 5,512,462, the disclosures of which are herein incorporated byreference. In representative PCR amplification reactions, the reactionmixture that includes the above extended nucleic acids or extensionproduct (which may also be viewed as a template nucleic acid in anamplification reaction) is combined with one or more primers that areemployed in the primer extension reaction, e.g., the PCR primers (suchas forward and reverse primers employed in geometric (or exponential)amplification or a single primer employed in a linear amplification).The oligonucleotide primers with which the template nucleic acid(hereinafter referred to as template DNA for convenience) is contactedwill be of sufficient length to provide for hybridization tocomplementary template DNA under annealing conditions (described ingreater detail below). The primers will generally be at least 10 bp inlength, usually at least 15 bp in length and more usually at least 16 bpin length and may be as long as 30 bp in length or longer, where thelength of the primers will generally range from 18 to 50 bp in length,usually from about 20 to 35 bp in length. The template DNA may becontacted with a single primer or a set of two primers (forward andreverse primers), depending on whether primer extension, linear orexponential amplification of the template DNA is desired.

As discussed above, the primers may be added to the sample before theaddition of the component comprising 3′ exonuclease activity, whereinsaid primers have been modified to be resistant to 3′ exonucleaseactivity, e.g. modification of the 3′ end. Furthermore, the primers mayalso be hot start primers, as described above.

In addition to the above components, the reaction mixture produced inthe subject methods typically includes a polymerase anddeoxyribonucleoside triphosphates (dNTPs). The desired polymeraseactivity may be provided by one or more distinct polymerase enzymes. Inmany embodiments, the reaction mixture includes at least a Family Apolymerase, where representative Family A polymerases of interestinclude, but are not limited to: Thermus aquaticus polymerases,including the naturally occurring polymerase (Taq) and derivatives andhomologues thereof, such as Klentaq (as described in Barnes et al, Proc.Natl. Acad. Sci USA (1994) 91:2216-2220) or iTaq (BioRad); Thermusthermophilus polymerases, including the naturally occurring polymerase(Tth) and derivatives and homologues thereof, and the like. In certainembodiments where the amplification reaction that is carried out is ahigh fidelity reaction, the reaction mixture may further include apolymerase enzyme having 3′-5′ exonuclease activity, e.g., as may beprovided by a Family B polymerase, where Family B polymerases ofinterest include, but are not limited to: Thermococcus litoralis DNApolymerase (Vent) as described in Perler et al., Proc. Natl. Acad. Sci.USA (1992) 89:5577-5581; Pyrococcus species GB-D (Deep Vent); Pyrococcusfuriosus DNA polymerase (Pfu) as described in Lundberg et al., Gene(1991) 108:1-6, Pyrococcus woesei (Pwo) and the like. Where the reactionmixture includes both a Family A and Family B polymerase, the Family Apolymerase may be present in the reaction mixture in an amount greaterthan the Family B polymerase, where the difference in activity willusually be at least 10-fold, and more usually at least about 100-fold.Usually the reaction mixture will include four different types of dNTPscorresponding to the four naturally occurring bases present, i.e. dATP,dTTP, dCTP and dGTP. In the subject methods, each dNTP will typically bepresent in an amount ranging from about 10 to 5000 μM, usually fromabout 20 to 1000 μM.

The reaction mixture prepared in this detection step of the subjectmethods may further include an aqueous buffer medium that includes asource of monovalent ions, a source of divalent cations and a bufferingagent. Any convenient source of monovalent ions, such as KCl, K-acetate,NH₄-acetate, K-glutamate, NH₄Cl, ammonium sulphate, and the like may beemployed. The divalent cation may be magnesium, manganese, zinc and thelike, where the cation will typically be magnesium. Any convenientsource of magnesium cation may be employed, including MgCl₂, Mg-acetate,and the like. The amount of Mg²⁺ present in the buffer may range from0.5 to 10 mM, but will preferably range from about 3 to 6 mM, and willideally be at about 5 mM. Representative buffering agents or salts thatmay be present in the buffer include Tris, Tricine, HEPES, MOPS and thelike, where the amount of buffering agent will typically range fromabout 5 to 150 mM, usually from about 10 to 100 mM, and more usuallyfrom about 20 to 50 mM, where in certain preferred embodiments thebuffering agent will be present in an amount sufficient to provide a pHranging from about 6.0 to 9.5, where most preferred is pH 7.3 at 72° C.Other agents which may be present in the buffer medium include chelatingagents, such as EDTA, EGTA and the like.

In preparing the reaction mixture of this step of the subject methods,the various constituent components may be combined in any convenientorder. For example, the buffer may be combined with primer, polymeraseand then template DNA, or all of the various constituent components maybe combined at the same time to produce the reaction mixture. In aparticularly preferred embodiment, the amplification reactants arecombined with the components for the extension and degradationreactions. In this respect, the components of the reaction arepreferably suitable for the activity all of the enzymatic components ofthe reaction, i.e. the component comprising 3′ exonuclease activity, the“first” polymerase for the production of the extension products andoptionally the “second” polymerase for the amplification step, which maybe different to the “first” polymerase. In some embodiments, the “first”and “second” polymerase are the same, i.e. the polymerase is capable ofextending the nucleic acid domain(s) of the proximity probe andamplifying at least a portion of the extended domain(s). In furtherembodiments, the polymerase comprises 3′ exonuclease activity. Forinstance, Phi29 (Φ29) DNA polymerase may be useful in embodiments wherethe product of the extension reaction is detected by RCA, i.e. Phi29(Φ29) DNA polymerase could be used as the 3′ exonuclease component, theextension component and the amplification component. A furthernon-limiting example is Pfu DNA polymerase, which may be useful inembodiments where the product of the extension reaction is detected byPCR, i.e. Pfu DNA polymerase could be used as the 3′ exonucleasecomponent, the extension component and the amplification component.

The amplified products of the amplification reaction may be detectedusing any convenient protocol, where the particular protocol employedmay detect the amplification products non-specifically or specifically,as described in greater detail below. Representative non-specificdetection protocols of interest include protocols that employ signalproducing systems that selectively detect double stranded DNA products,e.g., via intercalation. Representative detectable molecules that finduse in such embodiments include fluorescent nucleic acid stains, such asphenanthridinium dyes, including monomers or homo- or heterodimersthereof, that give an enhanced fluorescence when complexed with nucleicacids. Examples of phenanthridinium dyes include ethidium homodimer,ethidium bromide, propidium iodide, and other alkyl-substitutedphenanthridinium dyes. In another embodiment of the invention, thenucleic acid stain is or incorporates an acridine dye, or a homo- orheterodimer thereof, such as acridine orange, acridine homodimer,ethidium-acridine heterodimer, or 9-amino-6-chloro-2-methoxyacridine. Inyet another embodiment of the invention, the nucleic acid stain is anindole or imidazole dye, such as Hoechst 33258, Hoechst 33342, Hoechst34580 (BIOPROBES 34, Molecular Probes, Inc. Eugene, Oreg., (May 2000))DAPI (4′,6-diamidino-2-phenylindole) or DIPI(4′,6-(diimidazolin-2-yl)-2-phenylindole). Other permitted nucleic acidstains include, but are not limited to, 7-aminoactinomycin D,hydroxystilbamidine, LDS 751, selected psoralens (furocoumarins), styryldyes, metal complexes such as ruthenium complexes, and transition metalcomplexes (incorporating Tb³⁺ and Eu³⁺, for example). In certainembodiments of the invention, the nucleic acid stain is a cyanine dye ora homo- or heterodimer of a cyanine dye that gives an enhancedfluorescence when associated with nucleic acids. Any of the dyesdescribed in U.S. Pat. No. 4,883,867 to Lee (1989), U.S. Pat. No.5,582,977 to Yue et al. (1996), U.S. Pat. No. 5,321,130 to Yue et al.(1994), and U.S. Pat. No. 5,410,030 to Yue et al. (1995) (all fourpatents incorporated by reference) may be used, including nucleic acidstains commercially available under the trademarks TOTO, BOBO, POPO,YOYO, TO-PRO, BO-PRO, PO-PRO and YO-PRO from Molecular Probes, Inc.,Eugene, Oreg. Any of the dyes described in U.S. Pat. No. 5,436,134 toHaugland et al. (1995), U.S. Pat. No. 5,658,751 to Yue et al. (1997),and U.S. Pat. No. 5,863,753 to Haugland et al. (1999) (all three patentsincorporated by reference) may be used, including nucleic acid stainscommercially available under the trademarks SYBR Green, SYTO, SYTOX,PICOGREEN, OLIGREEN, and RIBOGREEN from Molecular Probes, Inc., Eugene,Oreg. In yet other embodiments of the invention, the nucleic acid stainis a monomeric, homodimeric or heterodimeric cyanine dye thatincorporates an aza- or polyazabenzazolium heterocycle, such as anazabenzoxazole, azabenzimidazole, or azabenzothiazole, that gives anenhanced fluorescence when associated with nucleic acids, includingnucleic acid stains commercially available under the trademarks SYTO,SYTOX, JOJO, JO-PRO, LOLO, LO-PRO from Molecular Probes, Inc., Eugene,Oreg. A further intercalating dye that may be of use in the methods ofthe invention is EvaGreen™ from Biotium Inc.

In a particularly preferred embodiment of the invention the extensionproduct is amplified by PCR, wherein the PCR is quantitative PCR and theamplified nucleic acid molecules are quantified using an intercalatingdye. In a preferred embodiment the intercalating dye is selected fromSYBR Green® and EvaGreen™.

In yet other embodiments, a signal producing system that is specific forthe amplification product, as opposed to double stranded molecules ingeneral, may be employed to detect the amplification. In theseembodiments, the signal producing system may include a probe nucleicacid that specifically binds to a sequence found in the amplificationproduct, where the probe nucleic acid may be labelled with a directly orindirectly detectable label. A directly detectable label is one that canbe directly detected without the use of additional reagents, while anindirectly detectable label is one that is detectable by employing oneor more additional reagents, e.g., where the label is a member of asignal producing system made up of two or more components. In manyembodiments, the label is a directly detectable label, where directlydetectable labels of interest include, but are not limited to:fluorescent labels, radioisotopic labels, chemiluminescent labels, andthe like. In many embodiments, the label is a fluorescent label, wherethe labelling reagent employed in such embodiments is a fluorescentlytagged nucleotide(s), e.g. fluorescently tagged CTP (such as Cy3-CTP,Cy5-CTP) etc. Fluorescent moieties which may be used to tag nucleotidesfor producing labelled probe nucleic acids include, but are not limitedto: fluorescein, the cyanine dyes, such as Cy3, Cy5, Alexa 555, Bodipy630/650, and the like. Other labels, such as those described above, mayalso be employed as are known in the art.

In certain embodiments, the specifically labelled probe nucleic acidsare labelled with “energy transfer” labels. As used herein, “energytransfer” refers to the process by which the fluorescence emission of afluorescent group is altered by a fluorescence-modifying group. If thefluorescence-modifying group is a quenching group, then the fluorescenceemission from the fluorescent group is attenuated (quenched). Energytransfer can occur through fluorescence resonance energy transfer, orthrough direct energy transfer. The exact energy transfer mechanisms inthese two cases are different. It is to be understood that any referenceto energy transfer in the instant application encompasses all of thesemechanistically-distinct phenomena. As used herein, “energy transferpair” refers to any two molecules that participate in energy transfer.Typically, one of the molecules acts as a fluorescent group, and theother acts as a fluorescence-modifying group. “Energy transfer pair” isused to refer to a group of molecules that form a single complex withinwhich energy transfer occurs. Such complexes may comprise, for example,two fluorescent groups which may be different from one another and onequenching group, two quenching groups and one fluorescent group, ormultiple fluorescent groups and multiple quenching groups. In caseswhere there are multiple fluorescent groups and/or multiple quenchinggroups, the individual groups may be different from one another. As usedherein, “fluorescence resonance energy transfer” or “FRET” refers to anenergy transfer phenomenon in which the light emitted by the excitedfluorescent group is absorbed at least partially by afluorescence-modifying group. If the fluorescence-modifying group is aquenching group, then that group can either radiate the absorbed lightas light of a different wavelength, or it can dissipate it as heat. FRETdepends on an overlap between the emission spectrum of the fluorescentgroup and the absorption spectrum of the quenching group. FRET alsodepends on the distance between the quenching group and the fluorescentgroup. Above a certain critical distance, the quenching group is unableto absorb the light emitted by the fluorescent group, or can do so onlypoorly. As used herein “direct energy transfer” refers to an energytransfer mechanism in which passage of a photon between the fluorescentgroup and the fluorescence-modifying group does not occur. Without beingbound by a single mechanism, it is believed that in direct energytransfer, the fluorescent group and the fluorescence-modifying groupinterfere with each others' electronic structure. If thefluorescence-modifying group is a quenching group, this will result inthe quenching group preventing the fluorescent group from even emittinglight.

The energy transfer labelled probe nucleic acid, e.g., oligonucleotide,may be structured in a variety of different ways, so long as it includesa donor, acceptor and target nucleic acid binding domains. As such, theenergy transfer labelled oligonucleotides employed in these embodimentsof the method are nucleic acid detectors that include a fluorophoredomain where the fluorescent energy donor, i.e., donor, is positionedand an acceptor domain where the fluorescent energy acceptor, i.e.,acceptor, is positioned. As mentioned above, the donor domain includesthe donor fluorophore. The donor fluorophore may be positioned anywherein the nucleic acid detector, but is typically present at the 5′terminus of the detector. The acceptor domain includes the fluorescenceenergy acceptor. The acceptor may be positioned anywhere in the acceptordomain, but is typically present at the 3′ terminus of the nucleic aciddetector or probe.

In addition to the fluorophore and acceptor domains, the energy transferlabelled probe oligonucleotides also include a target nucleic acidbinding domain, which binds to a target nucleic acid sequence found inthe amplification product of interest (as described above), e.g., understringent hybridization conditions (as defined above). This targetbinding domain typically ranges in length from about 10 to about 60nucleotides, usually from about 15 to about 30 nt. Depending on thenature of the oligonucleotide and the assay itself, the target bindingdomain may hybridize to a region of the template nucleic acid or aregion of the primer extension product. For example, where the assay isa 5′ nuclease assay, e.g., in which a TaqMan® type oligonucleotide probeis employed, the target binding domain hybridizes under stringentconditions to a target binding site of the template nucleic acid, whichis downstream or 3′ of the primer binding site. In alternativeembodiments, e.g., in molecular beacon type assays, the target bindingdomain hybridizes to a domain of a primer extension product. The overalllength of the energy transfer labelled oligonucleotides employed inthese embodiments, which includes all three domains mentioned above,typically ranges from about 10 to about 60 nucleotides, usually fromabout 15 to about 30 nucleotides.

In certain embodiments, the energy transfer labelled oligonucleotide isstructured such that energy transfer occurs between the fluorophore andacceptor of the energy transfer labelled oligonucleotide probe uponfluorophore excitation when the energy transfer labelled oligonucleotideis not hybridized to target nucleic acid.

In certain embodiments, the oligonucleotide is a single strandedmolecule that does not form intramolecular structures and in whichenergy transfer occurs because the spacing of the donor and acceptorprovides for energy transfer in the single stranded linear format. Inthese embodiments, energy transfer also occurs between the fluorophoreand acceptor of labelled oligonucleotide probe upon fluorophoreexcitation when the labelled oligonucleotide probe is hybridized to atarget nucleic acid. Specific examples of such labelled oligonucleotideprobes include the TaqMan® type probes, as described in U.S. Pat. No.6,248,526, the disclosure of which is herein incorporated by reference(as well as Held et al., Genome Res. (1996) 6:986-994; Holland et al.,Proc. Natl Acad. Sci. USA (1991) 88:7276-7280; and Lee et al., Nuc.Acids Res. (1993) 21:3761-3766). In many of these embodiments, thetarget nucleic acid binding domain is one that hybridizes to, i.e., iscomplementary to, a sequence of the template nucleic acid, i.e., thetarget nucleic acid of the target nucleic acid binding domain is asequence present in the template nucleic acid (i.e., the pseudotarget orsurrogate nucleic acid).

In other embodiments, the probe oligonucleotides are structured suchthat energy transfer does not occur between the fluorophore and acceptorof the energy transfer labelled oligonucleotide probe upon fluorophoreexcitation when the energy transfer labelled oligonucleotide probe ishybridized to a target nucleic acid. Examples of these types of probestructures include: Scorpion probes (as described in Whitcombe et al.,Nature Biotechnology (1999) 17:804-807; U.S. Pat. No. 6,326,145, thedisclosure of which is herein incorporated by reference), Sunrise probes(as described in Nazarenko et al., Nuc. Acids Res. (1997) 25:2516-2521;U.S. Pat. No. 6,117,635, the disclosure of which is herein incorporatedby reference), Molecular Beacons (Tyagi et al., Nature Biotechnology(1996) 14:303-308; U.S. Patent No. 5,989,823, the disclosure of which isincorporated herein by reference), and conformationally assisted probes(as described in provisional application Ser. No. 60/138,376, thedisclosure of which is herein incorporated by reference). In many ofthese embodiments, the target binding sequence or domain comprises ahybridization domain complementary to a sequence of the primer extensionproduct of the amplification reaction, and not to a sequence found inthe pseudotarget nucleic acid.

The next step in the subject methods is signal detection from thelabelled amplification products of interest, where signal detection mayvary depending on the particular signal producing system employed. Incertain embodiments, merely the presence or absence of detectablesignal, e.g., fluorescence, is determined and used in the subjectassays, e.g., to determine or identify the presence or absence of thetarget nucleic acid via detection of the pseudotarget nucleic acidand/or amplification products thereof. Depending on the particular labelemployed, detection of a signal may indicate the presence or absence ofthe target nucleic acid.

In those embodiments where the signal producing system is a fluorescentsignal producing system, signal detection typically includes detecting achange in a fluorescent signal from the reaction mixture to obtain anassay result. In other words, any modulation in the fluorescent signalgenerated by the reaction mixture is assessed. The change may be anincrease or decrease in fluorescence, depending on the nature of thelabel employed, but in certain embodiments is an increase influorescence. The sample may be screened for an increase in fluorescenceusing any convenient means, e.g., a suitable fluorimeter, such as athermostable-cuvette or plate-reader fluorimeter. Fluorescence issuitably monitored using a known fluorimeter. The signals from thesedevices, for instance in the form of photo-multiplier voltages, are sentto a data processor board and converted into a spectrum associated witheach sample tube. Multiple tubes, for example 96 tubes, can be assessedat the same time.

Where the detection protocol is a real time protocol, e.g., as employedin real time PCR reaction protocols, data may be collected in this wayat frequent intervals, for example once every 3 minutes, throughout thereaction. By monitoring the fluorescence of the reactive molecule fromthe sample during each cycle, the progress of the amplification reactioncan be monitored in various ways. For example, the data provided bymelting peaks can be analyzed, for example by calculating the area underthe melting peaks and these data plotted against the number of cycles.In a preferred embodiment of the invention, the fluorescence signal isachieved using a dye that intercalates in double stranded nucleic acidmolecules, preferably wherein the intercalating dye is selected fromSYBR Green® and EvaGreen™.

The spectra generated in this way can be resolved, for example, using“fits” of pre-selected fluorescent moieties such as dyes, to form peaksrepresentative of each signalling moiety (i.e. fluorophore). The areasunder the peaks can be determined which represents the intensity valuefor each signal, and if required, expressed as quotients of each other.The differential of signal intensities and/or ratios will allow changesin labelled probes to be recorded through the reaction or at differentreaction conditions, such as temperatures. The changes are related tothe binding phenomenon between the oligonucleotide probe and the targetsequence or degradation of the oligonucleotide probe bound to the targetsequence. The integral of the area under the differential peaks willallow intensity values for the label effects to be calculated.

Screening the mixture for a change in fluorescence provides one or moreassay results, depending on whether the sample is screened once at theend of the primer extension reaction, or multiple times, e.g., aftereach cycle, of an amplification reaction (e.g., as is done in real timePCR monitoring).

The data generated as described above can be interpreted in variousways. In its simplest form, an increase or decrease in fluorescence fromthe sample in the course of or at the end of the amplification reactionis indicative of an increase in the amount of the target analyte presentin the sample, e.g., as correlated to the amount of amplificationproduct detected in the reaction mixture, suggestive of the fact thatthe amplification reaction has proceeded and therefore the targetanalyte was in fact present in the initial sample. Quantification isalso possible by monitoring the amplification reaction throughout theamplification process. Quantification may also include assaying for oneor more nucleic acid controls in the reaction mixture, as describedabove.

In this manner, a reaction mixture may readily be screened (or assessedor assayed etc.) for the presence of target analyte(s). The methods aresuitable for detection of a single target analyte as well as multiplexanalyses, in which two or more different target analytes are assayed inthe sample. In these latter multiplex situations, the number ofdifferent sets of probes that may be employed typically ranges fromabout 2 to about 20 or higher, e.g., as up to 100 or higher, 1000 orhigher, etc.

The analysis of many analytes simultaneously and in a single reactionusing several different proximity probe sets (multiplexing) is madepossible by the increased specificity and sensitivity obtained with the3′ exonuclease method. Each probe set can be designed to produce aunique extension product that can be used to determine the presence orabsence, quantity and/or location of the analytes being interrogated bythe probe set. The extension product may be detected directly orpreferably after amplification using any of the well established methodsfor analysis of nucleic acid molecules known from the literatureincluding liquid chromatography, electrophoresis, mass spectrometry,microscopy, real-time PCR (quantitative PCR), fluorescent probes etc. Apreferred embodiment of the method of the invention utilisesquantitative or real-time PCR. Of particular interest is the combinationof the present method with a “DNA array” read-out format. Several uniqueextension products from a multiplexed proximity extension assay asdescribed herein may be hybridized to a standardized DNA array carryinga number of oligonucleotide sequences (tags) complementary to theextension product sequences. Each extension product hybridized to thearray may be identified by its location on the DNA array and thedetected intensity in a given hybridization spot will be indicative ofthe quantity of that specific extension product and hence also of theanalyte giving rise to that extension product. Detection of theextension products may be accomplished by spectrometry, fluorescence,radioisotopes etc. Fluorescent moieties may conveniently be introducedinto the extension products using fluorescently labelled primers orfluorescently labelled nucleotides in the amplification reaction (PCR).The DNA array may be a simple dot-blot array on a membrane containing asmall number of spots or a high density array carrying hundreds ofthousands of spots.

The method of the invention may be modified in order to further reducethe background associated with non-specific nucleic acid hybridizationevents. Such modifications include adjustments to the method that willreduce any non-specific nucleic acid hybridization events. In someembodiments, a protein may be added to the mixture containing the sampleand the proximity probes in order to reduce weak and non-specific DNAhybridization events. For example, E. coli single strand DNA bindingprotein has been used to increase the yield and specificity of primerextension reactions and PCR reactions. (U.S. Pat. Nos. 5,449,603 and5,534,407.) The gene 32 protein (single strand DNA binding protein) ofphage T4 apparently improves the ability to amplify larger DNA fragments(Schwartz, et al., Nucl. Acids Res. 18: 1079 (1990)) and enhances DNApolymerase fidelity (Huang, DNA Cell. Biol. 15: 589-594 (1996)). Whenemployed, such a protein will be used to achieve a concentration in thereaction mixture that ranges from about 0.01 ng/μL to about 1 μg/μL;such as from about 0.1 ng/μL to about 100 ng/μL; including from about 1ng/μL to about 10 ng/μL.

In some embodiments, the background may be reduced by the addition ofpoly-A RNA and/or bulk RNA in the assay. Bulk RNA is also known as totalRNA, i.e. bulk RNA is simply the total RNA extracted from a sample, e.g.a cell, comprising more than one form and preferably all of thedifferent forms of RNA present in said sample, e.g. mRNA, rRNA, microRNAetc.

In other embodiments, partially double stranded nucleic acid may be usedas the nucleic acid domain of the first and second proximity probes inorder to reduce weak and non-specific DNA hybridization events.

As explained above, the method of the invention is designed such thatinteraction between the nucleic acid domains of the first and secondprobes should occur only if the probes are bound to the analyte, becausethe nucleic acid domains of unbound probes with a free and unprotected3′ ends will be degraded fully or partially by the component comprising3′ exonuclease activity. However, as is the case with all assays of thistype, this cannot always be guaranteed and there may be some backgroundinteraction of the nucleic acid domains, if the probes come intoproximity randomly in solution (the possibility of this is reduced inembodiments that require the nucleic acid domains of the probes tohybridise to one another by means of the splint, in order for suchinteraction to occur; the chances of all three domains coming intoproximity randomly are reduced, compared to two-probe assays,nonetheless this may still under some circumstances occur). To reduce orminimise the possibility of background due to unbound (i.e. unreacted)probes, blocking oligonucleotides may be used in addition to any otherblocking reagents described above and known in the art. In a preferredembodiment the sample may be incubated with one or more blockingreagents, e.g. BSA and the like, prior to the addition of the proximityprobes.

The blocking oligonucleotides bind (i.e. hybridise or anneal) to thefree ends of the nucleic acid domains of the first and second proximityprobes. Thus a blocking oligonucleotide may bind to the free 3′ OH endof the nucleic acid domain of a 5′ proximity probe and to the free 5′phosphate end of the nucleic acid domain of a 3′ proximity probe. Thebinding of the blocking oligonucleotide may be out-competed in thepresence of a high local concentration of, e.g. a splintoligonucleotide, such as occurs in some embodiments of the invention. Inthis way the blocking oligonucleotide may prevent the first and seconddomains from hybridising to the splint in the absence of analytebinding. In other embodiments, one or more specific “competitor”oligonucleotides may be added to the assay, e.g. after the proximityprobes have become associated with the target analyte, to dissociate theblocking oligonucleotide from the ends of the nucleic acid domains ofthe probes and thereby allowing the domains of probes in proximity tointeract. Thus the free ends of the 5′ and/or 3′ probes may be preventedfrom interaction until after they have bound to the analyte. Inembodiments where a splint oligonucleotide is used and forms a nucleicacid domain of a third proximity probe, when all three probes are boundto the analyte, the local concentration of the splint is sufficient toout-compete the blocking oligonucleotides; the first and second domainshybridise to the splint and the blocking oligonucleotides are replaced.

The blocking oligonucleotides thus allow a competition-based strategy tobe used to reduce background and thus increase sensitivity of the assay.

The blocking oligonucleotides may range in length from about 4-100nucleotides, e.g. 6-75 or 10-50. They may hybridise to a region at ornear the free end of the nucleic acid domain of the first or secondprobe (“near” meaning within 1-20 or 1-10, e.g. 1-6 nucleotides of thefree 3′ or 5′ end). The region of hybridisation may be 3-15 nucleotideslong e.g. 3-12, 3-10, 3-8, 4-8, 3-6, 4-6.

The blocking oligonucleotides are typically used in an excess over therespective probes, e.g. an excess of 2-1000 fold, e.g. 20-500, 50-300,100-500, or 100-300 fold e.g., 20, 200 or 300 fold.

The competitor oligonucleotides are typically used in an excess over theblocking oligonucleotide, e.g. an excess of 2-1000 fold, e.g. 20-500,50-300, 100-500, or 100-300 fold e.g., 20, 200 or 300 fold.

In the case of detecting an analyte with proximity-probes of lowaffinity and slow binding kinetics, a preincubation step with theproximity-probes at a sufficiently high concentration promotes bindingof the proximity probes to the analyte. This preincubation step may bequickly diluted in a large volume of cold buffer (e.g., buffer that doesnot include the analyte or the proximity probes), and a portion of thisdilution subsequently added to a extension reaction mixture. The lowtemperature, e.g., ranging from about 0° C. to about 20° C., includingfrom about 4° C. to about 10° C., minimizes the dissociation of existingproximity-probe-analyte complexes while the vast dilution results in adecrease of the concentration of the unbound proximity-probes, therebylowering their reactivity and minimizing the background signal.

In such embodiments, the assay is performed by using a small incubationvolume of from about 1 μl to about 20 μl, such as about 1 μl, or about 2μl, or about 3 μl, or about 4 μl, or about 5 μl or about 6 μl, of sampleand proximity probes. The effective concentration of the proximityprobes in the final incubation volume is thus diluted, reducing thebackground while maintaining the signal since the binding between theprobes and analyte does not have time to dissociate before the first andthe second nucleic acid domains are extended. This approach enablesextremely high sensitivity as long as the extension products can beconcentrated from the larger volumes, such as over 100 μl or more, andthen detecting the proximity dependent interaction. In such embodiments,the probe-probe interactions can be reduced by using single strandbinding proteins.

Problems associated with complex samples may be addressed by dilutingthe complex sample prior to the analysis. This will greatly decrease theamount of proteins the probes may bind unspecifically to therebylowering concentration of probes required. While the analyte will alsobe diluted, the high sensitivity of the proximity probing will providegood detection and quantification.

The method of the present invention may be employed homogeneously (i.e.in solution) as described above, or alternatively heterogeneously, usinga solid phase, for example, in which bound analyte becomes immobilisedon a solid phase, permitting the use of washing steps. The use of solidphase assays offers advantages, particularly for the detection ofdifficult samples: washing steps can assist in the removal of inhibitingcomponents, and analytes can be enriched from an undesirably largesample volume. Higher concentrations and greater amounts of proximityprobes can be used, as unbound analytes and probes can be removed bywashing. The ability to remove unbound or unconjugated probes by washingalso means that the solid phase assay tolerates lower purity proximityprobes by comparison with the homogeneous assay.

Immobilisation of the analyte on a solid phase may be achieved invarious ways. Accordingly, several embodiments of the solid phase assayof the invention are contemplated. In one such embodiment, one (or more)of the first or second (or third proximity probes, if used) may be (ormay be capable of being) immobilised on a solid phase (or solidsupport). The analyte can firstly be captured by the one (or more)immobilised (or immobilisable) probes and secondly be bound bysubsequently added probe(s). In such a scheme, the previously-mentionedavidity effect may not be present during the binding step but isrelevant for the washing steps. Preferably, the analyte is contactedwith the solid phase-bound (i.e. immobilised, or immobilisable) probe(s)at the same time as the non-immobilised/non-immobilisable probe(s) areadded to the reaction mixture, such that the avidity effect contributesalso to the detection (binding) step.

The immobilised proximity probe may be immobilised, i.e. bound to thesupport, in any convenient way. Thus the manner or means ofimmobilisation and the solid support may be selected, according tochoice, from any number of immobilisation means and solid supports asare widely known in the art and described in the literature. Thus, theprobe may be directly bound to the support, for example via theanalyte-binding domain (e.g. chemically crosslinked), it may be boundindirectly by means of a linker group, or by an intermediary bindinggroup(s) (e.g. by means of a biotin-streptavidin interaction). Thus, aproximity probe may be provided with means for immobilisation (e.g. anaffinity binding partner, e.g. biotin or a hapten, capable of binding toits binding partner, i.e. a cognate binding partner, e.g. streptavidinor an antibody) provided on the support. The probe may be immobilisedbefore or after binding to the analyte. Further, such an “immobilisable”probe may be contacted with the sample together with the support.

The solid support may be any of the well known supports or matriceswhich are currently widely used or proposed for immobilisation,separation etc. These may take the form of particles (e.g. beads whichmay be magnetic or non-magnetic), sheets, gels, filters, membranes,fibres, capillaries, or microtitre strips, tubes, plates or wells etc.

The support may be made of glass, silica, latex or a polymeric material.Suitable are materials presenting a high surface area for binding of theanalyte. Such supports may have an irregular surface and may be forexample porous or particulate e.g. particles, fibres, webs, sinters orsieves. Particulate materials e.g. beads are useful due to their greaterbinding capacity, particularly polymeric beads.

Conveniently, a particulate solid support used according to theinvention will comprise spherical beads. The size of the beads is notcritical, but they may for example be of the order of diameter of atleast 1 and preferably at least 2 pm, and have a maximum diameter ofpreferably not more than 10, and e.g. not more than 6 μm.

Monodisperse particles, that is those which are substantially uniform insize (e.g. size having a diameter standard deviation of less than 5%)have the advantage that they provide very uniform reproducibility ofreaction. Representative monodisperse polymer particles may be producedby the technique described in US-A-4336173.

However, to aid manipulation and separation, magnetic beads areadvantageous. The term “magnetic” as used herein means that the supportis capable of having a magnetic moment imparted to it when placed in amagnetic field, e.g. the support may be para-magnetic, and thus isdisplaceable under the action of that field. In other words, a supportcomprising magnetic particles may readily be removed by magneticaggregation, which provides a quick, simple and efficient way ofseparating the particles following the analyte binding steps.

In another embodiment, an immobilised (or immobilisable)analyte-specific probe comprising only a binding domain (i.e. an analytecapture probe) can be used in addition to the non-immobilised proximityprobes of the homogeneous assay. Thus in such an embodiment the analyteis first captured by the immobilised or immobilisable capture probewhich serves only to immobilise the analyte on the solid phase, andsubsequently the immobilised analyte is incubated with the proximityprobes. In such an embodiment, the capture probe may be any bindingpartner capable of binding the analyte, directly or indirectly (e.g. asdiscussed above in relation to the analyte-binding domain of theproximity probe). More particularly, such a capture probe bindsspecifically to the analyte. Since this embodiment of the methodrequires the simultaneous binding of at least three probes (bindingdomains) to the analyte or analyte complex, potentially at least threedifferent epitopes can be interrogated, conferring high specificity onthe assay.

In a further embodiment, the analyte itself may be immobilised (orimmobilisable) on the solid phase e.g. by non-specific absorption. In aparticular such embodiment, the analyte may be present within cells,being optionally fixed and/or permeabilised, which are (capable ofbeing) attached to a solid support.

The above-described methods result in detection of splint-mediatedproximity dependent interactions that are present in the reactionmixture, which in turn provides a measure of the amount of targetanalyte in the sample being assayed. The measure may be qualitative orquantitative

Accordingly, the above described methods of detecting the presence ofone or more target analytes in a complex sample finds use in a varietyof different applications.

The subject methods may be used to screen a sample for the presence orabsence of one or more target analytes in a sample. As indicated above,the invention provides methods of detecting the presence or quantifyingthe amount of one or more target analytes in a sample.

The subject methods can be employed to detect the presence of one ormore target analytes in a variety of different types of samples,including complex samples having large amounts of non-target entities,where the subject methods provide for detection of the targetanalytes(s) with high sensitivity. As such, the subject methods arehighly sensitive methods of detecting one or more target analytes in asimple or complex sample. The sample that is assayed in the subjectmethods is, in many embodiments, from a physiological source, asdiscussed in more detail above.

In addition to detecting a wide variety of analytes, the subject methodsmay also be used to screen for compounds that modulate the interactionbetween the analyte binding domain of the proximity probe with thebinding region of the analyte i.e. the binding of the analyte-bindingdomain to the analyte. The term modulating includes both decreasing(e.g., inhibiting) and enhancing the interaction between the twomolecules. The screening method may be an in vitro or in vivo format,where both formats are readily developed by those of skill in the art.

A variety of different candidate agents may be screened by the abovemethods. Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Agents identified in the above screening assays find use in the avariety of methods, including methods of modulating the activity of thetarget analyte, and conditions related to the presence and/or activitythereof.

Also provided are kits that find use in practicing the subject methods,as described above. For example, in some embodiments, kits forpracticing the subject methods include at least one set of proximityprobes, which proximity probes each include an analyte binding domainand a nucleic acid domain as described above. As indicated above, thecertain protocols will employ two or more different sets of such probesfor simultaneous detection of two or more target analytes in a sample,e.g., in multiplex and/or high throughput formats. As such, in certainembodiments the kits will include two or more distinct sets of proximityprobes. Furthermore, additional reagents that are required or desired inthe protocol to be practiced with the kit components may be present,which additional reagents include, but are not limited to: a componentcomprising 3′ exonuclease activity, one or more polymerase enzymes,splint oligonucleotide (optionally in the form of a third proximityprobe), blocking oligonucleotides, competitor oligonucleotides, solidsupport for immobilisation of probe, binding domain or analyte, meansfor immobilisation of probe, binding domain or analyte, amplificationand detection means e.g. fluorescently labelled nucleotides oroligonucleotides or intercalating dyes (e.g. SYBR Green® and/orEvaGreen™), pairs of supplementary nucleic acids, single strand bindingproteins, and PCR amplification reagents (e.g., nucleotides, buffers,cations, etc.), and the like. In certain embodiments, the kits mayinclude elements employed in reducing the effective volume of anincubation mixture, as reviewed above, e.g., a volume excluder. The kitcomponents may be present in separate containers, or one or more of thecomponents may be present in the same container, where the containersmay be storage containers and/or containers that are employed during theassay for which the kit is designed.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, flash drive, etc., on which the information has beenrecorded. Yet another means that may be present is a website addresswhich may be used via the internet to access the information at aremoved site. Any convenient means may be present in the kits.

Accordingly, in a further aspect the present invention provides a kitfor use in a method for detecting an analyte in a sample, said kitcomprising:

(a) at least one set of at least first and second proximity probes,which probes each comprise an analyte-binding domain and a nucleic aciddomain and can simultaneously bind to the analyte; and

(b) a component comprising 3′ exonuclease activity; and

(c) optionally, means for extending the nucleic acid domain of at leastone of said first and second proximity probes to generate an extensionproduct; and

(d) optionally, means for amplifying and detecting said extensionproduct.

As indicated above, the means for extending the nucleic acid domains maybe a polymerase enzyme, and such means may optionally further comprisethe reagents necessary for the polymerase reaction (e.g. nucleotidesetc). The means for amplifying and detecting the extension product, maybe any of the means discussed above in the context of the assay methodse.g. amplification means and means for detecting amplification productsthereof e.g. reagents for a PCR reaction (e.g. amplification primers,and optionally polymerase and/or nucleotides, etc.) and for detectingPCR amplicons etc (e.g. Taqman® probes, intercalating dyes, such as SYBRGreen® and/or EvaGreen™, etc.).

The kit may further optionally comprise a splint oligonucleotide and/orblocking oligonucleotides for the first and second probes.

The kit may further optionally comprise an immobilised capture probe forthe analyte, or a capture probe provided with means for immobilisation.Alternatively, the kit may comprise a solid phase for capture of, orbinding to, the analyte, or one or more said first or second proximityprobes may be immobilised or provided with means for immobilisation.

The invention will be further described with reference to the followingnon-limiting Examples with reference to the following drawings in which:

FIG. 1 shows a schematic representation of five different versions ofproximity extension assays.

FIG. 2 is a bar chart showing the activity of various polymerases with3′ exonuclease activity or in combination with a 3′ exonuclease in thedetection of interleukin-8 (50 pM) using the methods of the invention.

FIG. 3 shows a graph that plots the signal generated from the detectionof an analyte (the ICAM antigen) in a sample using the methods of theinvention in the presence and absence of a “crowding agent”, sephadexG-100.

FIG. 4 shows graph that plots the signal generated from the detection ofan analyte (phycoerythrin, PE) in a sample using the methods of theinvention, where the analyte was added to either buffer or plasma.

FIG. 5 shows graph that plots the signal generated from the detection ofan analyte (Glial-cell Derived Neurotrophic Factor, GDNF) in a sampleusing the methods of the invention, where the analyte was added tobuffer or naturally present in plasma.

FIG. 6 shows graph that plots the signal generated from the detection ofan analyte (Interleukin-8, IL8) in a sample using the “one step” methodof the invention.

FIG. 7 shows graph that plots the signal generated from the detection ofan analyte (Vascular endothelial growth factor, VEGF) in a sample usingthe “one step” method of the invention.

FIG. 8 shows a bar chart showing the activity of two hyperthermophilicpolymerases with 3′ exonuclease activity, namely Pfu DNA polymerase andPwo DNA Polymerase (labelled as Hypernova™, the commercially availablepolymerase from DNA GDAŃSK) in the detection of interleukin-8 (100 pM)using the methods of the invention.

EXAMPLES

Experimental Procedure, Detection of Interleukin-8 (IL8)

Proximity-Probe Preparation

One batch of polyclonal antibody (RnD Systems, AF-208-NA) was coupledwith Innovas Lightning-Link conjugation technology to two differentssDNA strands, one attached with the 5′-end:

(SEQ ID NO: 1) 5′-GGCCCAAGTGTTAATTTGCTTCACGATGAGACTGGATGAA-3′

and the second one at the 3′-end:

(SEQ ID NO: 2) 5′-TCACGGTAGCATAAGGTGCAGTATGAGAACTTCGCTACAG-3′

A third oligonucleotide, referred to as “Extension oligo”, was added tothe 3′-attached conjugates at a 2:1 (oligo:conjugate) ratio.

PEA Protocol #1 (Two Step Version)

1 μL sample (PBS+0.1% BSA buffer, IL-8 antigen standard from RnD Systems208-IL-010, EDTA plasma) was mixed with 1 μL blocking buffer containing0.21 mg/ml goat IgG (Sigma Aldrich 19140), 107 μg/ml single strandedsalmon sperm DNA (Sigma Aldrich D7656), 0.085% BSA, 4.3 mM EDTA, 0.21%Triton-X100, 0.02% sodium azide and 2.5 μM blocking conjugates (OlinkAB, WO 2012/007511). Samples were blocked at 25° C. for 20 minutes.

To 2 μLs of the blocked samples 2 μL of probe mix (25 mM Tris-HCl, 4 mMEDTA, 1 mM Biotin, 0.016 mg/ml single stranded salmon sperm DNA (SigmaAldrich D7656), 0.02% sodium azide and 100 pM of each PEA conjugate) wasadded and then incubated at 37° C. for 1 hour.

After the probe incubation the samples were transferred to a thermalcycler and put on hold at 37° C. 76 μL of dilution mix containing 70.5mM Tris-HCl, 17.7 mM ammonium sulfate, 1.05 mM dithiothreitol and 40 μM(each) of dNTP's were added to the incubated samples. After 5 minutes at37° C. the second addition of 20 μL extension mix (66.8 mM Tris-HCl,16.8 mM ammonium sulfate, 1 mM dithiothreitol, 33 mM magnesium chlorideand 62.5 U/mL T4 DNA Polymerase (Fermentas, #EP0062)), or other DNApolymerases, were added. The extension reactions were performed at 37°C. for another 20 minutes and then heat inactivated at 80° C. for 10minutes.

For the qPCR detection of extension products, 4 μL of the extensionproducts were transferred to a qPCR plate and mixed with 6μL qPCR mix(25 mM Tris-HCl, 7.5 mM magnesium chloride, 50 mM potassium chloride,8.3 mM ammonium sulfate, 8.3% Trehalose (Acros Organics, 182550250), 333μM (each) dNTP's, 1.67 mM dithiothreitol, 833 nM of each primer(forward: 5′-TCGTGAGCCCAAGTGTTAATTTGCTTCACGA-3′ (SEQ ID NO: 3), reverse:5′-TGCAGTCTGTAGCGAAGTTCTCATACTGCA-3′ (SEQ ID NO: 4), Biomers), 417 nMMolecular Beacon (FAM-CCCGCTCGCTTATGCTACCGTGACCTGCGAATCCCGAGCGGG-DABSYL,(SEQ ID NO: 5) Biomers), 41.7 U/mL recombinant Taq polymerase (Fermentas#EP0402) and 1.33 μM ROX reference (ROX-TTTTTTT, Biomers). Two step qPCRwas run with initial denaturation at 95° C. for 5 minutes, followed by45 cycles of 95° C. denaturation for 15 seconds and 60° C. combinedannealing and extension for 1 minute.

Comparison of Different DNA Polymerases

When comparing a sample of DNA polymerases that have 3′->5′ exonucleaseactivity (T4 DNA Polymerase, DNA Polymerase, Phi29 DNA Polymerase, DNAPolymerase I, Klenow Fragment) with those without (Klenow Fragmentexo(−)), it was found there is a distinct difference in signal relativeto the background, see FIG. 2. The Klenow Fragment without 3′exonuclease activity has a much higher background than the KlenowFrament (3′ exo+), and if Exonuclease I is added to the Klenow Fragmentexo(−) similar results to those using the Klenow fragment (3′ exo+) wereacheived. So exonuclease I, which is not a DNA polymerase, can rescuethe reaction polymerized by Klenow (exo−).

Some DNA polymerases containing 3′ exonuclease activity, e.g. T7 andphi-29 DNA polymerase, gave low signals both for background and antigensamples (FIG. 2). It is hypothesised that this result is a consequenceof the strong 3′ exonuclease activity found in these enzymes and furtheroptimization of the specific reaction conditions for these enzymes wouldenable these enzymes to find utility in the methods described herein.

The positive effect of using an enzyme comprising 3′ exonucleaseactivity in proximity extension assays is assumed arise from degradationof the free non-proximal DNA ends so that they can not accumulateextension products over time during the extension reactions itself.

Probe Incubation with Sephadex G-100 Beads

In order to investigate the possibility of enhancing PEA performance bythe use of molecular crowding agents, e.g. crowding polymers, assuggested in US 20090162840 for proximity ligation assays, the use ofsephadex beads was tested using the above described methods. Drysephadex beads can upon rehydration in solution expand and by absorbingwater, whilst larger molecules such as proteins (i.e. analytes) andproximity probes that may be present in the solution remain outside thebeads. This effectively enhances the concentration of said proteins andproximity probes, thereby promoting target binding by said probes. FIG.3 shows the results of an assay wherein 0.5 μg of sephadex G-100 waslyophilized at the bottom of the PEA incubation tubes onto which theprobe mix and sample was added. Dramatic improvements in the sensitivityand signal to noise ratio are seen, indicating increased target binding.

Plasma Recovery and Matrix Effects on PEA

To test the ability of PEA using 3′ exonuclease efficient DNApolymerases to accurately detect proteins in a complex matrix, the assaydescribed above were performed using human EDTA prepared plasma. Thenon-human protein phycoerythrin (PE) was spiked at variousconcentrations into either a non-complex matrix PBS (phosphate bufferedsaline) with 0.1% BSA, or a very complex matrix EDTA plasma andquantified by PEA. A non-human protein was selected so that recoverycould be assessed even at low concentrations. Excellent recovery wasobserved for this analyte in plasma even at 10 pM (FIG. 4) as there wasvirtually no difference in signal between complex and non-complexmatrix.

In another example, the human protein GDNF (Glial-cell DerivedNeurotrophic Factor) was spiked into human plasma, FIG. 5. This proteinsis of very low abundance in plasma and difficult to detect with standardtechnologies. Again, outstanding recovery was observed for this analytedemonstrating that endogenous GDNF signal can be quantified at below 0.1pM.

Simplified PEA Protocol (#2)

If there is a possibility to simplify experimental procedures, it isalways desirable to do so. It was found that it was possible to combinethe extension mix with the qPCR mix, making it possible to directlytransfer the extension products from the thermal cycler to the qPCRinstrument without any further pipetting and with less hands-on time.This protocol proved to retain a high sensitivity and signal along withgood precision (FIGS. 6 and 7). In order to enable the use of a 3′exonuclease DNA polymerase for the proximity extension reaction in thepresence of the qPCR primers, it was necessary to make said primersresistant to exonucleases. This was achieved by producing primers thatform a hairpin structure at the lower temperature of the PEA reactionand modifying the last two residues at the 3′-end with phosphothioate.The one step version of the PEA protocol is set out below. The resultsfor 1L8 and VEGF detection are set out in FIGS. 6 and 7, respectivelyand obtained using the one step protocol.

PEA Protocol #2 (One Step Version)

1 μL sample (PBS+0.1% BSA buffer, IL-8 antigen standard from RnD Systems208-IL-010, EDTA plasma) was mixed with 1 μL blocking buffer containing0.19 mg/ml goat IgG (Sigma Aldrich 19140), 94 μg/ml single strandedsalmon sperm DNA (Sigma Aldrich D7656), 0.075% BSA, 3.8 mM EDTA, 0.19%Triton-X100, 0.015% sodium azide and 2.5 μM blocking conjugates (OlinkAB, WO 2012/007511). Samples were blocked at 25° C. for 20 minutes.

To 2 μLs of the blocked samples, 2 μL of probe mix (25 mM Tris-HCl, 4 mMEDTA, 1 mM Biotin, 0.016 mg/ml single stranded salmon sperm DNA (SigmaAldrich D7656), 0.02% sodium azide and 100 pM of each PEA conjugate) wasadded and then incubated at 37° C. for 1 hour.

Following the probe incubation step, the samples were transferred to athermal cycler and put on hold at 37° C. 36 μL of a dilution mixcontaining 1× iTaq SYBR Green Supermix (BioRad, 172-5851) and 3′-thioateprotected hairpin primers (forward:5′-TCGTGAGCCCAAGTGTTAATTTGCTTCAC*G*A-3′ (SEQ ID NO: 6), reverse:TGCAGTCTGTAGCGAAGTTCTCATACTG*C*A-3′ (SEQ ID NO: 7), * indicates thioatemodifications) was added to the samples. After 3 minutes at 37° C., 10μL of extension mix (1× iTaq SYBR Green Supermix (BioRad, 172-5851) and62.5 U/mL T4 DNA Polymerase (Fermentas, #EP0062)) was added. Theextension reactions were performed at 37° C. for another 20 minutes andthen the T4 DNA polymerase was heat inactivated at 65° C. for 10minutes.

The reaction mix (50 μL) contained the extension product and all of thereagents needed for qPCR. 10 μL of the reaction mix was transferred toan optical qPCR plate for quantification. Two step qPCR was run withinitial denaturation at 95° C. for 5 minutes, followed by 45 cycles of95° C. denaturation for 15 seconds and 64° C. combined annealing andextension for 1 minute.

1.-2. (canceled)
 3. A method for detecting an analyte in a sample,comprising: (a) contacting said sample with one set or multiple sets ofat least first and second proximity probes, which probes each comprisean analyte-binding domain and a nucleic acid domain and simultaneouslybind to the analyte; (b) allowing the nucleic acid domains of theproximity probes to interact with each other upon binding of saidproximity probes to said analyte, wherein said interaction comprises theformation of a duplex; (c) contacting said sample with a componentcomprising 3′ exonuclease activity, wherein said component comprises apolymerase enzyme (c1) having 3′ exonuclease activity and/or anexonuclease enzyme (c2) other than the polymerase enzyme (c1), (d)extending the 3′ end of at least one nucleic acid domain of said duplexto generate an extension product, wherein the step may occurcontemporaneously with or after step (c) and wherein the extension isperformed by said polymerase (c1), or by a separate polymerase enzymewhich is added during or after step (c); (e) amplifying the extensionproduct; and (f) detecting the amplified extension product; whereineither: (i) the nucleic acid domains of the proximity probes aresingle-stranded and interact directly via regions of complementarity toeach other; or (ii) the nucleic acid domain of at least one of theproximity probes is partially double-stranded and comprises asingle-stranded domain hybridised to a splint oligonucleotide, and thesplint oligonucleotide is extended in step (d).
 4. The method of claim3, wherein the analyte is a wholly or partially proteinaceous molecule.5. The method of claim 3, wherein the analyte binding domain of at leastone of said at least first and second proximity probes is an antibody,or a binding fragment thereof or derivative thereof.
 6. The method ofclaim 3, wherein the polymerase (c1) having 3′ exonuclease activitycomprises T4 DNA polymerase, T7 DNA polymerase, Phi29 (Φ29) DNApolymerase, DNA polymerase I, Klenow fragment of DNA polymerase I,Pyrococcus furiosus (Pfu) DNA polymerase, Pyrococcus woesei (Pwo) DNApolymerase, and/or an RNA polymerase.
 7. The method of claim 3, whereinstep (d) is carried out by an exo-nucleic acid polymerase with no orminimal 3′ exonuclease activity.
 8. The method of claim 7, wherein theexo-polymerase is added after the exonuclease enzyme (c2), wherein thesample is further incubated to allow the extension products to begenerated and/or wherein the exo-polymerase is selected from the asubunit of DNA polymerase Ill, the Klenow exo(−) fragment of DNApolymerase I, Taq polymerase, Pfu (exo−) DNA polymerase and/or Pwo(exo−) DNA polymerase.
 9. The method of claim 3, wherein theamplification of step (e) is performed using a different polymerase thanthe polymerase enzyme (c1) having 3′ exonuclease activity that is usedin step (d) for extension.
 10. The method of claim 3, wherein thecomponent comprising 3′ exonuclease activity is exonuclease I or an RNApolymerase and/or wherein the component comprising 3′ exonucleaseactivity is inactivated prior to the step of amplifying the extensionproduct.
 11. The method of claim 10, wherein the 3′ exonuclease activityis inactivated by heat denaturation.
 12. The method of claim 11, whereinthe heat inactivation is a first step of the amplification reaction ofstep (e).
 13. The method of claim 3, wherein amplifying the extensionproduct comprises binding an amplification reagent to the extensionproduct, wherein the amplification reagent is selected from the groupconsisting of: (i) a primer for the amplification; (ii) a padlock probeor circular oligonucleotide comprising a sequence which is complementaryto an extended part of the extension product; and (iii) a templateoligonucleotide which acts as a ligation template for circularization ofan oligonucleotide comprising an extended part of the extension productthereby to provide a template for amplification; and wherein theamplification reagent is added before, at the same time or after thecontacting step (c).
 14. The method of claim 13, wherein when thenucleic acid domain of at least one of the proximity probes is partiallydouble-stranded and comprises a single-stranded domain hybridised to asplint oligonucleotide, and the splint oligonucleotide is extended, thetemplate oligonucleotide of part (iii) binds to the extended splintoligonucleotide.
 15. The method of claim 13, wherein the amplificationis rolling circle amplification.
 16. The method of claim 3, wherein step(e) comprises amplifying a portion of the extended part of the extensionproduct.
 17. The method of claim 16, wherein amplifying a portion of theextended part of the extension product is achieved using a first primerand a second primer, such that the primers flank, respectively, sides ofthe portion of the extended part of the extension product.
 18. Themethod of claim 16, wherein the portion of the extended part of theextension product (i) templates the ligation of an oligonucleotide togenerate a circular oligonucleotide, wherein the circularoligonucleotide acts as a template for amplification; or (ii) acts as aprimer for rolling circle amplification of a circular oligonucleotide.19. The method of claim 18, wherein the amplification of part (i) isrolling circle amplification.
 20. The method of claim 3, wherein theamplification comprises a polymerase chain reaction.
 21. The method ofclaim 20, wherein the polymerase chain reaction is a quantitativepolymerase chain reaction.
 22. The method of claim 21, wherein thequantitative polymerase chain reaction uses a dye which intercalateswith nucleic acid molecules to provide a detectable signal.
 23. Themethod of claim 22, wherein the dye which intercalates with nucleic acidmolecules to provide a detectable signal is SYBR Green® or EvaGreen™.24. The method of claim 20, wherein the primers used in the polymerasechain reaction are provided in a modified form such that they areresistant to 3′ exonuclease activity.
 25. The method of claim 24,wherein the primers comprise at least one modified nucleotide at the 3′end.
 26. The method of claim 25, wherein the modified nucleotide isselected from any one or more of the group consisting of athiophosphate-modified nucleotide, a locked nucleic acid nucleotide, a2′-OMe-CE Phosphoramidite modified nucleotide, and a peptide nucleicacid nucleotide.
 27. The method of claim 20, wherein primers for thepolymerase chain reaction are hotstart primers and/or wherein at leastone of the polymerases is a thermostable polymerase.
 28. The method ofclaim 3, wherein both of the nucleic acid domains of the first andsecond proximity probes are extended in step (d).
 29. The method ofclaim 3, wherein the splint oligonucleotide of part (ii) ispre-hybridised to the nucleic acid domain of said proximity probe. 30.The method of claim 3, wherein the splint oligonucleotide of part (ii)is provided separately as a free nucleic acid molecule.
 31. The methodof claim 30, wherein the splint oligonucleotide is added to the samplebefore, at the same time as, or after the proximity probes.
 32. Themethod of claim 3, wherein the splint oligonucleotide of part (ii) isprovided as the nucleic acid domain of a third proximity probe.
 33. Themethod of claim 32, wherein the third proximity probe is added to thesample at the same time as the first and second proximity probes. 34.The method of claim 3, comprising multiplex analysis for detecting twoor more analytes in a sample, said method comprising contacting thesample with multiple sets of at least first and second proximity probes,wherein each of the sets binds to one of the two or more analytes, andthe nucleic acid domains of each set of the proximity probes interactwith each other upon binding of said proximity probes to a respectiveanalyte to form a duplex; extending the 3′ end of at least one nucleicacid domain of each of the duplexes and generating a unique extensionproduct for each of the sets; amplifying each of the unique extensionproducts; and detecting the amplified unique extension products.
 35. Themethod of claim 3, wherein a crowding agent is included in step (a)and/or (b).
 36. The method of claim 35, wherein the crowding agent issephadex, preferably wherein the sephadex is type G-100.
 37. A kit foruse in a method for detecting an analyte in a sample, said kitcomprising: a) one set or multiple sets of first and second proximityprobes, which probes each comprise an analyte-binding domain and anucleic acid domain and simultaneously bind to the analyte; whereineither: i) the nucleic acid domains of the proximity probes aresingle-stranded and interact directly via regions of complementarity toeach other; or ii) the nucleic acid domain of at least one of theproximity probes is partially double-stranded and comprises asingle-stranded domain hybridised to a splint oligonucleotide; and b) anenzyme comprising 3′ exonuclease activity, wherein said enzyme is a 3′exonuclease enzyme other than a polymerase enzyme. c) optionally apolymerase enzyme; d) optionally means for amplifying and detecting saidextension product; e) optionally a splint oligonucleotide and/orblocking oligonucleotides for the first and second probes; and f)optionally an immobilised capture probe for the analyte, or a captureprobe provided with means for immobilisation.